Compositions including beta-hydroxy-beta-methylbutyrate and uses thereof

ABSTRACT

The present invention relates to methods for the prevention and treatment of chronic inflammatory diseases, cancer, and involuntary weight loss. In the practice of the present invention patients are enterally administered HMB alone or alternatively in combination with eicosapentaenoic (20:5 ω-3), FOS, carnitine and mixtures thereof. HMB may be added to food products comprising a source of amino-nitrogen enriched with large neutral amino acids such as leucine, isoleucine, valine, tyrosine, threonine and phenylalanine and substantially lacking in free amino acids.

This application is a divisional of prior U.S. application Ser. No.11/025,466 filed Dec. 29, 2004, which is a continuation-in-part of priorU.S. application Ser. No. 10/810,762 filed Mar. 26, 2004, the entiredisclosures of which are incorporated herein by reference.

The present invention relates to methods for the prevention andtreatment of chronic inflammatory diseases, cancer, and involuntaryweight loss. In the practice of the present invention patients areenterally administered HMB alone or alternatively in combination witheicosapentaenoic (20:5 ω-3), FOS, carnitine and mixtures thereof. HMBmay be added to food products comprising a source of amino-nitrogenenriched with large neutral amino acids such as leucine, isoleucine,valine, tyrosine, threonine and phenylalanine and substantially lackingin free amino acids.

BACKGROUND

Undesired weight loss, particularly lean mass loss is a relativelycommon occurrence in critical illness, and has a significant impact onmorbidity and mortality. This is particularly true in cancer patients,where such mass losses can become treatment-limiting, and thus impactoverall prognosis.

Cachexia is a syndrome characterized by anorexia, weight loss, prematuresatiety, asthenia, loss of lean body mass, and multiple organdysfunction. It is a common consequence of chronic illnesses (bothmalignant and non-malignant) and is associated with a poorer prognosisin chronic obstructive pulmonary disease (COPD), chronic heart failure(CHF), renal failure, AIDS, dementia, chronic liver disease and cancer.It is often independent of other indicators of disease severity. (Witte,K. K. A. and Clark, A. L.: Nutritional abnormalities contributing tocachexia in chronic illness, International Journal of Cardiology85:23-31, 2002)

Pulmonary disease is often associated with cachexia, and substantialnumbers of patients suffering from COPD, particularly emphysema, becomeemaciated during the course of the disease. Weight loss is anindependent risk factor for prognosis, and is often associated withincreased oxygen consumption. COPD is also associated with a generalelevated systemic inflammatory response, reflected by elevatedconcentrations of pro-inflammatory cytokines and acute phase proteins inthe peripheral blood. Such changes are often associated with musclewasting syndromes.

Studies with incubated muscles and muscle extracts suggest that theATP-dependent ubiquitin-proteosome pathway is responsible for most ofthe increased proteolysis which ultimately results in muscle wasting. Inparticular, increased levels of ubiquitin-conjugated proteins, andincreases in mRNA levels for polyubiquitin, certain proteosome subunitsand the ubiquitin-conjugating enzyme E2_(14K) are features found in mostatrophying muscles.

The majority of patients with cancer whose disease progresses tometastatic disease develop cachexia during their treatment program andthe cachexia contributes to their deaths. The frequency of weight lossin cancer patients ranges from 40% for patients with breast cancer,acute myelocytic leukemia, and sarcoma to more than 80% in patients withcarcinoma of the pancreas and stomach. About 60% of patients withcarcinomas of the lung, colon or prostate have experienced weight lossprior to beginning chemotherapy. Although the relationship betweenpretreatment malnutrition (weight loss) and adverse outcome isestablished, no consistent relationship has been demonstrated betweenthe development of cachexia and tumor size, disease stage, and type orduration of the malignancy.

Cancer cachexia is not simply a local effect of the tumor. Alterationsin protein, fat, and carbohydrate metabolism occur commonly. Forexample, abnormalities in carbohydrate metabolism include increasedrates of total glucose turnover, increased hepatic gluconeogenesis,glucose intolerance and elevated glucose levels. Increased lipolysis,increased free fatty acid and glycerol turnover, hyperlipidemia, andreduced lipoprotein lipase activity are frequently noted. The weightloss associated with cancer cachexia is caused not only by a reductionin body fat stores but also by a reduction in total body protein mass,with extensive skeletal muscle wasting. Increased protein turnover andpoorly regulated amino acid oxidation may also be important. Thepresence of host-derived factors produced in response to the cancer havebeen implicated as causative agents of cachexia, e.g., tumor necrosisfactor-α (TNF) or cachectin, interleukin-1 (IL-1), IL-6,gamma-interferon (IFN), and prostaglandins (PGs) (e.g., PGE₂).

Weight loss is common in patients with carcinomas of the lung andgastrointestinal tract, resulting in a massive loss of both body fat andmuscle protein, while non-muscle protein remains unaffected. While lossof body fat is important in terms of energy reserves, it is loss ofskeletal muscle protein that results in immobility, and eventuallyimpairment of respiratory muscle function, leading to death fromhypostatic pneumonia. Although cachexia is frequently accompanied byanorexia, nutritional supplementation alone is unable to maintain stablebody weight and any weight that is gained is due to an increase inadipose tissue and water rather than lean body mass. The same is truefor appetite stimulants, such as megestrol acetate andmedroxyprogesterone acetate, suggesting that loss of lean body mass isdue to factors other than energy insufficiency.

Skeletal muscle mass is a balance between the rate of protein synthesisand the rate of degradation. Patients with cancer cachexia show adepression of protein synthesis in skeletal muscle and an increase inprotein degradation, which is reflected in an increased expression ofthe ubiquitin-proteasome proteolytic pathway, the major determinant ofprotein degradation. Thus skeletal muscle from cachectic cancer patientsshows increased expression of mRNA for both ubiquitin and proteasomesubunits, while proteasome proteolytic activity increased in parallelwith ubiquitin expression. The inability of anabolic stimuli to increaselean body mass in cachectic patients suggests that protein degradationmust be attenuated before muscle mass can increase. Eicosapentaenoicacid (EPA), downregulates the increased expression of theubiquitin-proteasome proteolytic pathway in the skeletal muscle ofcachectic mice, and has been shown to stabilize body weight in cachecticpatients with pancreatic cancer. When patients consumed an energy-densesupplement containing 32 g protein and 2 g EPA body weight increased andthis was attributed solely to an increase in lean body mass (Barber, M.D., Ross, J. A., Voss, A. C., Tisdale, M. J., Fearon, K. C. H. Theeffect of an oral nutritional supplement enriched with fish oil onweight-loss in patients with pancreatic cancer. Br. J. Cancer, 81:80-86, 1999).

A recent study by May et al (May, P. E., Barber, A., D'Olimpio, J. T.,Hourihane, A. and Abumrad, N. N. Reversal of cancer-related wastingusing oral supplementation with a combination ofβ-hydroxy-β-methylbutyrate, arginine and glutamine. Am. J. Surg., 183:471-479, 2002) showed a mixture of HMB, arginine and glutamine to beeffective in increasing body weight in weight losing patients withadvanced (stage IV) cancer. Moreover, the increase in body weight wasattributed to an increase in fat-free mass, as observed with EPA.

The use of the polyunsaturated fatty acid eicosapentaenoic acid issuggested for the treatment of cachexia by inhibiting lipolytic activityof lipolytic agents in body fluids and the activity of the enzymeguanidino-benzoatase. See Tisdale, M. J., and Beck, A., U.S. Pat. No.5,457,130, issued Oct. 10, 1995; and Tisdale, et al. Cancer Research 50:5022-5026 (August 1990). However, the product taught by Tisdale was in asolid dosage form, requiring an already ill patient to swallow 12-16capsules per day. This method had serious drawbacks, includingdifficulty in swallowing, belching, and bad odor.

HMB has been found to be useful within the context of a variety ofapplications. Specifically, in U.S. Pat. No. 6,031,000 to Nissen et al.describes a composition comprising HMB, free L-arginine, and freeL-glutamine. This patent also provides a method for the treatment ofdisease-associated wasting of an animal and other methods comprisingadministering to the animal a composition comprising HMB and at leastone free amino acid.

U.S. Pat. No. 5,348,979 to Nissen et al. describes the use of HMB in thenitrogen retention in human subjects. The amount of HMB administered iseffective to conserve protein as determined by reduction in urinarynitrogen. The method can be used with patients having a negativenitrogen balance due to disease conditions, and also with normal elderlypersons who are subject to protein loss.

U.S. Pat. No. 5,028,440 to Nissen describes a method for raising meatproducing domestic animals to increase lean tissue development. HMB isfed within the range of from 0.5 to 100 mg.

U.S. Pat. No. 4,992,470 to Nissen describes the use of HMB to bemarkedly more effective for activating the immune function of Tlymphocytes of mammals than .alpha.-ketoisocaproate (KIC). Foractivation of the T lymphocytes, HMB or an edible water-soluble saltthereof is administered to the mammal by a route through which the HMBenters the blood of the mammal. The amount administered is sufficientfor effective enhancement of the blastogenesis of their T lymphocytes.

Some bodybuilding advertising claims make the bare assertion that HMBpromotes protein synthesis, (see, e.g. websites:

http://www.bodybuilding.com/store/kzn/hmb.html;http://www.interactivenutrition.com/products/hmb.php; andhttp://www.interactivenutrition.com/learningzone/hmb.php )but these lack any scientific documentation and amount to mere “puffery”that appears to misstate the established inhibitory effect of HMBprotein degradation, which also leads to gain in muscle mass but is not“synthesis”. The only scientific study making this suggestion is a 1996abstract by Ostaszewski, et al (J. Anim. Sci 1996; 74(Supppl 1)); whichclaims their data in rats and chicks indicates that “HMB stimulates[protein synthesis] slightly (avg. 6%) and markedly decreased [proteinbreakdown] (avg. −18%)”. A later paper by 4 of the same authors usingthe same model in rats and chicks failed to repeat the synthesis effectand concludes that “HMB had no significant effect on protein synthesis”(Ostaszewski, et al (J. Anim. Physiol. a. Anim. Nutr. 84 (2000), 1-8).This leaves doubt and uncertainty about whether HMB stimulates proteinsynthesis or not, but in any event, each of these authors reports onnormal subjects; not one addresses the effect of HMB in individualswhose muscle status is compromised by a disease-associated wastingcondition. In such conditions, protein synthesis is significantlydepressed.

German patent DE 29707308 to Kunz describes the use of branched chainamino acids in combination with HMB to promote muscle generation in theweight training population. Kunz teaches that a supplement of 3 gm takendaily with a protein consumption of 200 gm per day enhances the value ofnutritional protein and significantly increases the protein efficiency.Kunz also teaches that better effects can be achieved when HMB iscombined with protein hydrolysates and/or free amino acid mixturesrather than with intact (pure) proteins.

U.S. Pat. No. 5,976,550 to Engel et al. describes a dietary foodsupplement for weight reduction formed of a mixture of a sugar basedconfectionary containing therapeutic amounts of chitosan, kava and a fatburning nutriceutical which may include choline/inusital, chromiumpicolinate, HMB, carnitine and pyruvate. The nutriceutical ingredientmixed with the chitosan and kava functions to burn whatever fat the bodyhas consumed, i.e. to metabolize better any fat that is ingested and notattracted to the chitosan.

Commercial products designed for the weight lifting population thatcontain HMB include Lean DynamX by EAS Inc. of Golden, Colo. Lean DynamXprovides a blend of ingredients that support fat loss without the use ofstrong stimulants. The ingredients include HMB, chromium picolinate,conjugated linoleic acid, mate leaves and stems and carnitine tartrate.The powder composition is mixed with water and taken 2-3 servings daily,with one serving taken 30 minutes before workouts.

Additional commercial products include Mega HMB Fuel® from TwinlabCorporation in Hauppauge, N.Y. Mega HMB Fuel® contains 750 mg of HMB inone capsule. The suggested daily dosage is 4 capsules to support damageto muscle cells which can occur subsequent to intense resistanceexercise.

Also of interest is U.S. Pat. No. 5,444,054 to Garleb, et al. andrelated U.S. Pat. Nos. 5,780,451 and 6,468,987. These documents describecompositions and methods useful in the treatment of ulcerative colitis.Such compositions include a protein source that can be intact orhydrolyzed proteins of high biological value (col. 21); an indigestibleoligosaccharide such as fructooligosaccharide; and a lipid blendcontaining a relatively high proportion of eicosapentaneoic acid, whichcontributes to a relatively high ω-3 to ω-6 fatty acid ratio.

Long chain fatty acid bio-pathways and physiological actions arediscussed in U.S. Pat. No. 5,223,285 to DeMichele, et al., the entiretyof which is incorporated herein by reference.

The prevention and/or treatment of cachexia remain a frustratingproblem. Both animal and human studies suggest that nutritional supportis largely ineffective in repleting lean body mass in the cancer-bearinghost. Randomized trials exploring the usefulness of total parenteralnutrition (TPN) support as an adjunct to cytotoxic antineoplastictherapy have demonstrated little improvement in treatment results. Seefor example Brennan, M. F., and Burt, M. E., 1981, Cancer TreatmentReports 65 (Suppl. 5): 67-68. This, along with a clear demonstrationthat TPN can stimulate tumor growth in animals suggests the routine useof TPN in cancer treatment is not justified. Kisner, D. L., 1981, CancerTreatment Reports 65 (Suppl. 5): 1-2.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention relates to methods for theprevention and treatment of chronic inflammatory diseases, cancer,and/or involuntary weight loss. In the practice of the present inventionpatients are enterally administered HMB alone or alternatively incombination with one or more of eicosapentaenoic acid (EPA) (20:5 ω-3),FOS, large neutral amino acids (LNAA), carnitine and mixtures thereof.

In another embodiment, the present invention provides a method for thetreatment of the disease-associated wasting of a patient. The methodcomprises administering to the patient the above-described composition,which comprises HMB in amounts sufficient to treat thedisease-associated wasting, wherein, upon administration of thecomposition to the patient, the disease-associated wasting is treated.This effect is produced in part by the known inhibition of proteinbreakdown, and in part by the surprising finding that protein synthesisis significantly stimulated by HMB in wasting patients.

In another embodiment, the present invention provides a method forreducing tumor growth rate in a patient. The method comprisesadministering to the patient the above-described composition, whichcomprises HMB in amounts sufficient to reduce tumor growth rate,wherein, upon administration of the composition to the patient, thetumor growth rate is reduced.

In another embodiment, the present invention provides a method for theprevention or treatment of diseases in patients by down regulating theexpression and/or activity of protein kinase C, nuclear factor kappa-B,ubiquitin-conjugating enzymes, and components of 26S proteasome. Thesemethods comprise administering to the patient HMB, its salts,metabolites or derivatives thereof.

In yet another embodiment, the present invention relates tocompositions, for example nutritional compositions, containing HMB aloneor alternatively in combination with one or more of eicosapentaenoicacid (20:5 ω-3), FOS, large neutral amino acids (LNAA), carnitine andmixtures thereof. HMB may be added to food products comprising a sourceof amino-nitrogen enriched with large neutral amino acids such asleucine, isoleucine, valine, tyrosine, threonine and phenylalanine andsubstantially lacking in free amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a scheme describing the potential intracellular eventsin skeletal muscle involved in PIF induced proteasome activation.

FIG. 2 presents dose-response curves for the effect of HMB on bodyweight (A) and tumor volume (B) in mice bearing the MAC16 tumor. HMB (inPBS) was administered orally by gavage on a daily regime at aconcentration of 0.05 (), 0.125 (◯) and 0.25 glkg (X). Control micereceived PBS alone (♦). The results shown are the mean±SEM, where n=20.

FIG. 3 presents the effect of HMB (0.25 glkg; ▪), EPA (0.6 glkg; X) andthe combination (◯) together with PBS controls () on body weight ofmice bearing the MAC16 tumor. Results shown are mean±SEM, where n=20.

FIG. 4 presents the weight of soleus muscles (A) and rate of proteindegradation in soleus muscle (B) of mice bearing the MAC16 tumor andtreated with either EPA (0.6 g/kg), HMB (0.25 g/kg) or the combinationfor 3 days. Values shown are mean±SEM, where n=6.

FIG. 5 presents the effect of HMB and EPA on proteasome functionalactivity, determined as the ‘chymotrypsin-like’ enzyme activity, ingastrocnemius muscle of mice bearing the MAC16 tumor and treated for 3days. Results are shown as mean±SEM, where n=6.

FIG. 6 presents the expression of proteasome 20S α-subunits (A) andβ-subunits (B), detected by Western blotting, in gastrocnemius muscle ofmice treated for 3 days with PBS (Control), HMB (0.25 g/kg), EPA (0.6g/kg) or the combination. Densitometric analysis of the blots (n=6) areshown. A. control (closed bars), HMB (open bars), EPA (hashed bars) andcombination (dotted bars)

FIG. 7 presents the expression of proteasome 19S subunits, MSS1 (A) andp42 (B), detected by Western blotting, in gastrocnemius muscle of micetreated for 3 days with PBS (Control), HMB (0.25 glkg), EPA (0.6 glkg)or the combination (HMB+EPA). Densitometric analysis of the blots (n=6)are shown.

FIG. 8 presents the expression of E2_(14k), detected by Westernblotting, in gastrocnemius muscle of mice treated for 3 days with PBS(Control), HMB (0.25 glkg), EPA (0.6 glkg) or the combination (HMB+EPA).Densitometric analysis of the blots (n=6) are shown.

FIG. 9 (A) presents the effect of PIF on total protein degradation inC₂C₁₂ myotubes in the absence (X) or presence of either 50 μM EPA (□),or 25 μM (O) or 50 μM () HMB. Measurements were made 24 h after theaddition of PIF and are shown as mean±SEM, where n=9. 1(B) presents thechymotryptic activity of soluble extracts of murine myotubes treatedwith PIF in the absence or presence of EPA (50 μM) or HMB (25 or 50 μM).The symbols are the same as in (A). The results are shown as mean±SEM,where n=9.

FIG. 10 presents the effect of EPA and HMB on PIF-induction of 20Sproteasome α-subunit (A), β-subunit (B) and p42 (C). The actin loadingcontrol is shown in (D). Western blots of soluble extracts of C₂C₁₂myotubes 24 h after treatment with PIF alone (lanes A-C) or with PIF inthe presence of 50 μM EPA (lanes D-F), 50 μM HMB (lanes G-I) or 25 μMHMB (lanes J-L) at a concentration of PIF of 4.2 nM (lanes B, E, H andK) or 10 nM (lanes C, F, I and L). Control cultures received PBS (laneA), 50 μM EPA (lane D), 50 μM HMB (lane G) or 25 μM HMB (lane J). Theblots shown are representative of three separate experiments.

FIG. 11 presents the Western blot of the effect of PIF on cytoplasmic(A) and membrane-bound (B) PKC_(α) in murine myotubes. Cells weretreated with PIF alone (lanes A-C) or with PIF in the presence of 50 μMEPA (lanes D-F), 50 μM HMB (lanes G-I) or 25 μM HMB (lanes J-L) at 4.2nM (lanes B, E, H and K) or 10 nM PIF (lanes C, F, I and L). Controlcells received PBS (lane A), 50 μM EPA (lane D), 50 μM HMB (lane G) or25 μM HMB (lane J). The blots shown are representative of three separateexperiments.

FIG. 12 presents Western blots of total ERK ½ (p44 and p42) (A) andactive (phosphorylated) ERK ½ (B) in soluble extracts of murine myotubestreated with PIF alone (lanes A-C) or with PIF in the presence of 50 μMEPA (lanes D-F), 50 μM HMB (lanes G-I) or 25 μM HMB (lanes J-L) at a PIFconcentration of 4.2 nM (lanes B, E, H and K) or 10 nM (lanes C, F, Iand L). Control cells received either PBS (lane A), 50 μM EPA (lane D),50 μM HMB (lane G) or 25 μM HMB (lane J). The blots shown arerepresentative of three separate experiments.

FIG. 13 presents the effect of exposure of C₂C₁₂ myotubes for 30 min oncytosolic levels of IκBα (A), determined by Western blotting, andactivation of NF-κB binding to DNA, as determined by EMSA (B and C). Thedensitometric analysis is an average of 3 replicate blots or EMSAs. (A)Myotubes were treated with PIF alone (lanes A-E) or with PIF in thepresence of 50 μM HMB at a concentration of 0 (lanes A and F), 2.1(lanes B and G), 4.2 (lanes C and H), 10.5 (lanes D and I) or 16.8 nMPIF (lanes E and J). In (B) and (C) myotubes were treated with 0, 2.1,4.2, 10.5 or 16.8 nM PIF, in the absence (dark bars) or presence (openbars) of 25 μM HMB (B) or 50 μM HMB (C).

FIG. 14 presents the effects of HMB on protein synthesis (stimulated)and degradation (inhibited) in the gastrocnemius muscles of MAC16tumor-bearing animals. Panel 14 A shows the effect of daily p.o.administration of 0.25 g/kg and 2.5 g/kg HMB on the rate of proteinsynthesis (dark-shaded columns) and degradation (unshaded columns),expressed as millimoles of phenylalanine incorporated or released pergram of muscle per 2 hours. Treatment was terminated after 3 days.Columns, mean (n=6); bars, SE. Differences from PBS control: a, P<0.05;b, P<0.001. Panel 14 B shows the ratio of the rate of protein synthesisto the rate of protein degradation in gastrocnemius muscles of micetreated in A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term HMB, which is also referred to asbeta-hydroxy-beta-methylbutyric acid, or beta-hydroxy-isovaleric acid,can be represented in its free acid form as (CH₃)₂(OH)CCH₂ COOH. HMB isa metabolite of leucine formed by transamination toalpha-ketoisocaproate (KIC) in muscle followed by oxidation of the KICin the cytosol of the liver to give HMB. While any suitable form of HMBcan be used within the context of the present invention, preferably, HMBis selected from the group consisting of a free acid, a salt, an ester,and a lactone; more preferably, HMB is in the form of a non-toxic,edible salt. Preferably, the HMB salt is water-soluble or becomeswater-soluble in the stomach or intestines of a patient. Morepreferably, the HMB salt is selected from the group consisting of asodium salt, a potassium salt, a magnesium salt, a chromium salt, and acalcium salt. However, other non-toxic salts, such as other alkali metalor alkaline earth metal salts, can be used.

Similarly, any pharmaceutically acceptable ester can be used in thecontext of the present invention. Desirably, the HMB ester is rapidlyconverted to HMB in its free acid form. Preferably, the HMB ester is amethyl ester or ethyl ester. HMB methyl ester and HMB ethyl ester arerapidly converted to the free acid form of HMB. Likewise, anypharmaceutically acceptable lactone can be used in the context of thepresent invention. Desirably, the HMB lactone is rapidly converted toHMB in its free acid form. Preferably, the HMB lactone is an isovalaryllactone or a similar lactone. Such lactones are rapidly converted to thefree acid form of HMB.

Methods for producing HMB and its derivatives are well known in the art.For example, HMB can be synthesized by oxidation of diacetone alcohol.One suitable procedure is described by Coffman et al., J. Am. Chem. Soc.80: 2882-2887 (1958). As described therein, HMB is synthesized by analkaline sodium hypochlorite oxidation of diacetone alcohol. The productis recovered in free acid form, which can be converted to the desiredsalt. For example, 3-hydroxy-3-methylbutyric acid (HMBA) can besynthesized from diacetone alcohol (4-hydroxy-4-methylpentan-2-one) viaoxidation using cold, aqueous hypochlorite (bleach). After acidifyingthe reaction mixture using HCl, the HMBA product is recovered byextraction using ethyl acetate, and separating and retaining the organiclayer from the extraction mixture. The ethyl acetate is removed byevaporation and the residue dissolved in ethanol. After addition ofCa(OH)₂ and cooling, crystalline CaHMB can be recovered by filtration,the crystals washed with ethanol and then dried. Alternatively, thecalcium salt of HMB is commercially available from Technical SourcingInternational (TSI) of Salt Lake City, Utah.

The term “eicosapentanoic acid” or “EPA” refers to the long chain,polyunsaturated fatty acid designated in the art as (20:5 ω-3),described further herein.

The term “large neutral amino acids” refers to leucine, isoleucine,valine, tyrosine, threonine and phenylalanine. Amino acids are thebuilding blocks of proteins. They are characterized by the presence of acarboxyl group (COOH) and an amino group (NH2) attached to the samecarbon at the end of the compound.

The term “substantially lacking in free amino acids” refers tocompositions which contain less than 0.4 grams of total free amino acidcontent in a daily dose of the composition. For example, if the productis designed to be fed at the rate of 1 can per day, then the one can ofproduct contains less than a total of 0.4 grams of free amino acids. Theamino acids in question are those naturally occurring L-isomers,consisting of one or more of the following compounds: L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-cysteine (or L-cystine),L-glutamic acid, L-glutamine, glycine, L-histidine, L-Isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine and L-valine, or their food- orpharmaceutically-acceptable salts, esters, salts or derivatives (such asmethyl or ethyl esters).

The term “cachexia” refers to a state of general ill health andmalnutrition. It is often associated with and induced by malignantcancer, and is characterized by loss of appetite, loss of body mass,especially lean body mass, and muscle wasting.

The term “fatty acids” refer to a family of carboxylic acids having ahydrocarbon chain, generally from about 12 to 24 carbons long. Whenunsaturated (having a double bond) at least one point in the hydrocarbonchain, such fatty acids are designated by the position of the firstdouble bond. ω-3 fatty acids have a first double bond at the thirdcarbon from the methyl end of the chain; and include, but are notlimited to, a-linolenic acid, stearidonic acid, eicosapentaenoic acid(“EPA”), docosapentaenoic acid and docosahexaenoic acid (“DHA”) and thelike. ω-6 fatty acids have a first double bond at the sixth carbon fromthe methyl end of the chain; and include, but are not limited to,linoleic acid, γ-linolenic acid, arachidonic acid (“AA”), and the like.

The term “food products” as used herein refer to delivery vehicles thatcontain one or more of fats, amino nitrogen and carbohydrates andprovides some or all of the nutritional support for a patient in therecommended daily amounts. Frequently a food product will containvitamins, minerals, trace minerals and the like to provide balancednutrition to meal replacements, medical foods, supplements. The foodproducts may be in any typical form such as beverages, powders, bars,juices, carbonated beverages, bottled water.

The term “Reference Daily Intakes or RDI” refers to a set of dietaryreferences based on the Recommended Dietary Allowances for essentialvitamins and minerals. The Recommended Dietary Allowances are a set ofestimated nutrient allowances established by the National Academy ofSciences, which are updated periodically to reflect current scientificknowledge.

The term “patient” refers to humans, dogs, cats, and any othernon-ruminant animal.

Any reference to a numerical range in this application should beconsidered as being modified by the adjective “about”. Further, anynumerical range should be considered to provide support for a claimdirected to a subset of that range. For example, a disclosure of a rangeof from 1 to 10 should be considered to provide support in thespecification and claims to any subset in that range (i.e., ranges of2-9, 3-6, 4-5, 2.2-3.6, 2.1-9.9, etc.).

Administration:

Nutritional support in the cancer patient can be categorized as (i)supportive, in which nutrition support is instituted to preventnutrition deterioration in the adequately nourished patient or torehabilitate the depleted patient before definitive therapy; (ii)adjunctive, in which nutrition support plays an integral role in thetherapeutic plan; and (iii) definitive, in which aggressive nutritionsupport is required for the patient's existence. The routes forproviding nutrition support include an oral diet, tube feeding andperipheral or total parenteral nutrition. The preferred embodiment fornutritional methods and compositions of the invention is by the oralroute.

An alternate to oral feeding is tube feeding by means of nasogastric,nasoduodenal, esophagostomy, gastrostomy, or jejunostomy tubes.

The beneficial effects that HMB has on the lean body mass of a patientcan be achieved in a number of ways. If desired, the HMB may beadministered alone, without a carrier. The HMB may simply be dissolvedin water and consumed by the patient. Alternatively, the HMB may besprinkled on food, dissolved in coffee, etc. The total daily dose forthe patient will vary widely, but typically a patient will benefit fromconsuming at least 2 gm/day of HMB. Alternatively, from 20 to 40mg/kg/day.

In a further embodiment, the HMB may be incorporated into pills,capsules, rapidly dissolved tablets, lozenges, etc. The active dose canvary widely, but will typically range from 250 mg to 1 gm/dose with thepatient consuming from 2 to 8 doses/day to achieve the target of 2gm/day minimum. Methods for preparing such dosage forms are well knownin the art. The reader's attention is directed to the most recentedition of Remingtons Pharmaceutical Sciences for guidance on how toprepare such dosage forms.

Nutritional Matrices:

While the HMB may be administered as a single entity, it will typicallybe incorporated into food products and consumed by the patient duringtheir meals or snack. If desired, the patient may simply modify therecipe of foods they normally consume by sprinkling on food, dissolvingin coffee, etc.

In a further embodiment, the HMB will be incorporated into beverages,bars, cookies, etc. that have been specifically designed to enhance thepalatability of the HMB and increase the selection of alternative forms,thereby enhancing patient/consumer acceptance.

Typically, the HMB will be incorporated into meal replacement beveragessuch as Ensure®, Boost®, Glucerna®, Pediasure®, Pedialyte®, etc. The HMBmay also be incorporated into meal replacement bars such as PowerBars®,Glucerna® bars, Choice DM® bars, Ensure® bars, and Boost® bars, etc.Alternatively, the HMB maybe incorporated into juices, carbonatedbeverages, bottled water, etc. Additionally, the HMB may be incorporatedinto medical nutritionals such as ProSure®, Promote®, Jevity® andAdvera® designed to support specific disease states such as cancer,HIV/AIDS, COPD arthritis, etc. Methods for producing any of such foodproducts are well known to those skilled in the art. The followingdiscussion is intended to illustrate such food products and theirpreparation.

Most meal replacement products (i.e., bars or liquids) provide caloriesfrom fat, carbohydrates, and protein. These products also typicallycontain vitamins and minerals, because they are intended to be suitablefor use as the sole source of nutrition. While these meal replacementproducts may serve as the sole source of nutrition, they typicallydon't. Individuals consume these products to replace one or two meals aday, or to provide a healthy snack. The nutritional products of thisinvention should be construed to include any of these embodiments.

The amount of these nutritional ingredients can vary widely dependingupon the targeted patient population (i.e., cancer, HIV/AIDS, arthritis,organoleptic considerations, cultural preferences, use, etc.). As ageneral nonlimiting guideline however, the meal replacement products ofthis invention will contain the following relative amounts of protein,fat, and carbohydrate (based upon the relative percentage of totalcalories): a protein component, providing from 5 to 80% of the totalcaloric content, a carbohydrate component providing from 10 to 70% ofthe total caloric content, and a lipid component providing from 5 to 50%of the total caloric content.

The meal replacements will contain suitable carbohydrates, lipids andproteins as is known to those skilled in the art of making nutritionalformulas. Suitable carbohydrates include, but are not limited to,hydrolyzed, intact, naturally and/or chemically modified starchessourced from corn, tapioca, rice or potato in waxy or non waxy forms;and sugars such as glucose, fructose, lactose, sucrose, maltose, highfructose corn syrup, corn syrup solids, fructooligosaccharides, andmixtures thereof.

Suitable lipids include, but are not limited to, coconut oil, soy oil,corn oil, olive oil, safflower oil, high oleic safflower oil, MCT oil(medium chain triglycerides), sunflower oil, high oleic sunflower oil,palm oil, palm olein, canola oil, cottonseed oil, fish oil, palm kerneloil, menhaden oil, soybean oil, lecithin, lipid sources of arachidonicacid and docosahexaneoic acid, and mixtures thereof. Lipid sources ofarachidonic acid and docosahexaneoic acid include, but are not limitedto, marine oil, egg yolk oil, and fungal or algal oil.

Numerous commercial sources for these fats are readily available andknown to one practicing the art. For example, soy and canola oils areavailable from Archer Daniels Midland of Decatur, Ill. Corn, coconut,palm and palm kernel oils are available from Premier Edible OilsCorporation of Portland, Oreg. Fractionated coconut oil is availablefrom Henkel Corporation of LaGrange, Ill. High oleic safflower and higholeic sunflower oils are available from SVO Specialty Products ofEastlake, Ohio. Marine oil is available from Mochida International ofTokyo, Japan. Olive oil is available from Anglia Oils of NorthHumberside, United Kingdom. Sunflower and cottonseed oils are availablefrom Cargil of Minneapolis, Minn. Safflower oil is available fromCalifornia Oils Corporation of Richmond, Calif.

In addition to these food grade oils, structured lipids may beincorporated into the food product if desired. Structured lipids areknown in the art. A concise description of structured lipids can befound in INFORM, Vol. 8, No. 10, page 1004; entitled Structured lipidsallow fat tailoring (October 1997). Also see U.S. Pat. No. 4,871,768.Structured lipids are predominantly triacylglycerols containing mixturesof medium and long chain fatty acids on the same glycerol nucleus.Structured lipids and their use in enteral formula are also described inU.S. Pat. Nos. 6,194,379 and 6,160,007.

Optionally, ω-3 fatty acids may comprise up to approximately 30% of theoil blend, preferably the ω-3 fatty acids largely consist of the longerchain forms, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).Dietary oils used in the preparation of the nutritional compositiongenerally contain ω-3 fatty acids in the triglyceride form and include,but are not limited to canola, medium chain triglycerides, fish,soybean, soy lecithin, corn, safflower, sunflower, high-oleic sunflower,high-oleic safflower, olive, borage, black currant, evening primrose andflaxseed oil. Optionally, the weight ratio of ω-6 fatty acids to ω-3fatty acids in the lipid blend according to the invention is about 0.1to 3.0. The daily delivery of ω-3 fatty acids should be at least 450 mgand may vary depending on body weight, sex, age and medical condition ofthe individual. As mentioned, higher levels are desired for adult humanconsumption: for example, from about 0.5 to 50 gm daily, more preferablyfrom about 2.5 to 5 gm daily.

An unexpected advantage to combining ω-3 fatty acids and HMB is theimprovement in taste of the meal replacement. The typical sources of ω-3fatty acids are fish and algae oils. Each source brings objectionableflavors to the meal replacement product. The Inventors discovered thatby adding HMB, the same or better clinical results related to theprevention of involuntary weight loss can be obtained even when usingsub-optimal or lower levels of ω-3 fatty acids in the product.Consequently, the Inventor's have discovered that there is an inverserelationship between the levels of ω-3 fatty acids and HMB. For example,if an effective does of ω-3 fatty acids is 3 gm delivered in 2 cans of ameal replacement, the same clinical results would be seen in productformulated to contain 2 gm of ω-3 fatty acids and 1 gm of HMB deliveredin 2 cans or in product formulated to contain 1 gm of ω-3 fatty acidsand 2 gm of HMB delivered in 2 cans. The product formulated to containonly 1 gm of ω-3 fatty acids will taste much better than the productformulated with 2 or 3 gm of ω-3 fatty acids while achieving the sameclinical effectiveness. Further, since ω-3 fatty acids are knowninhibitors of AA, a mediator of inflammation, a product containing ω-3fatty acids and HMB could have broader benefits than those containingeither of the ingredients alone.

Suitable protein sources include, but not limited to, milk, whey andwhey fractions, soy, rice, meat (e.g., beef), animal and vegetable(e.g., pea, potato), egg (egg albumin), gelatin and fish. Suitableintact protein sources include, but are not limited to, soy based, milkbased, casein protein, whey protein, rice protein, beef collagen, peaprotein, potato protein, and mixtures thereof.

Optionally, the intact protein source is enriched in large neutral aminoacids (LNAA) comprising valine, isoleucine, leucine, threonine, tyrosineand phenylalanine. Typically, about 40% of casein, whey and soy proteinsources are large neutral amino acids. For example, caseinate containsabout 38 wt/wt % LNAA, whey protein concentrate contains about 39 wt/wt% LNAA and soy protein isolate contains about 34 wt/wt % LNAA.Typically, the meal replacement is formulated with a protein source thatwill deliver about 1 to 25 gm of LNAA per day, preferably from about 1to 20 gm of LNAA per day, more preferably from about 4 to 20 gm of LNAAper day. As an example, a meal replacement consumed 3 times a day thatcontains a protein comprising 4.8 gm LNAA will deliver 14.4 gm LNAA perday.

The meal replacements preferably also contain vitamins and minerals inan amount designed to supply or supplement the daily nutritionalrequirements of the person receiving the formula. Those skilled in theart recognize that nutritional formulas often include overages ofcertain vitamins and minerals to ensure that they meet targeted levelover the shelf life of the product. These same individuals alsorecognize that certain micro ingredients may have potential benefits forpeople depending upon any underlying illness or disease that the patientis afflicted with. For example, cancer patients benefit from suchantioxidants as beta-carotene, vitamin E, vitamin C and selenium. Thefood products preferably include, but are not limited to, the followingvitamins and minerals: calcium, phosphorus, sodium, chloride, magnesium,manganese, iron, copper, zinc, selenium, iodine, chromium, molybdenum,conditionally essential nutrients m-inositol, carnitine and taurine, andVitamins A, C, D, E, K and the B complex, and mixtures thereof.

The conditionally essential nutrient carnitine is a naturally occurringamino acid formed from methionine and lysine. Its major metabolic roleis associated with the transport of long-chain fatty acids across themitochondrial membranes, thus stimulating the oxidation of these fuelsubstances for metabolic energy. Carnitine supplementation is animportant metabolic tool in conditions such as diseases of the liver andkidney, and major chronic illnesses or extensive injuries complicated bymalnutrition. Optionally, the meal replacements may be supplemented withcarnitine at levels sufficient to supply up to 4 gm/day of carnitine.

The meal replacements also may contain fiber and stabilizers. Suitablesources of fiber/and or stabilizers include, but are not limited to,xanthan gum, guar gum, gum arabic, gum ghatti, gum karaya, gumtracacanth, agar, furcellaran, gellan gum, locust bean gum, pectin, lowand high methoxy pectin, oat and barley glucans, carrageenans, psyllium,gelatin, microcrystalline cellulose, CMC (sodiumcarboxymethylcellulose), methylcellulose hydroxypropyl methyl cellulose,hydroxypropyl cellulose, DATEM (diacetyl tartaric acid esters of mono-and diglycerides), dextran, carrageenans, FOS (fructooligosaccharides),and mixtures thereof. Numerous commercial sources of soluble dietaryfibers are available. For example, gum arabic, hydrolyzedcarboxymethylcellulose, guar gum, pectin and the low and high methoxypectins are available from TIC Gums, Inc. of Belcamp, Md. The oat andbarley glucans are available from Mountain Lake Specialty Ingredients,Inc. of Omaha, Nebr. Psyllium is available from the Meer Corporation ofNorth Bergen, N.J. while the carrageenan is available from FMCCorporation of Philadelphia, Pa.

The fiber incorporated may also be an insoluble dietary fiberrepresentative examples of which include oat hull fiber, pea hull fiber,soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose andcorn bran. Numerous sources for the insoluble dietary fibers are alsoavailable. For example, the corn bran is available from Quaker Oats ofChicago, Ill.; oat hull fiber from Canadian Harvest of Cambridge, Minn.;pea hull fiber from Woodstone Foods of Winnipeg, Canada; soy hull fiberand oat hull fiber from The Fibrad Group of LaVale, Md.; soy cotyledonfiber from Protein Technologies International of St. Louis, Mo.; sugarbeet fiber from Delta Fiber Foods of Minneapolis, Minn. and cellulosefrom the James River Corp. of Saddle Brook, N.J.

A more detailed discussion of examples of fibers and their incorporationinto food products may be found in U.S. Pat. No. 5,085,883 issued toGarleb et al.

The quantity of fiber utilized in the formulas can vary. The particulartype of fiber that is utilized is not critical. Any fiber suitable forhuman consumption and that is stable in the matrix of a food product maybe utilized.

In addition to fiber, the meal replacements may also containoligosaccharides such as fructooligosaccharides (FOS) orglucooligosaccharides (GOS). Oligosaccharides are rapidly andextensively fermented to short chain fatty acids by anaerobicmicroorganisms that inhabit the large bowel. These oligosaccharides arepreferential energy sources for most Bifidobacterium species, but arenot utilized by potentially pathogenic organisms such as Clostridiumperfingens, C. difficile, or Eschericia coli.

Typically, the FOS comprises from 0 to 5 gm/serving of the mealreplacement, preferably from 1 to 5 gm/serving, more preferably from 2to 4 gm/serving of the meal replacement.

The meal replacements may also contain a flavor to enhance itspalatability. Artificial sweeteners may be added to complement theflavor and mask salty taste. Useful artificial sweeteners includesaccharin, nutrasweet, sucralose, acesulfane-K (ace-K), etc.

Meal replacements can be manufactured using techniques well known tothose skilled in the art. Various processing techniques exist. Typicallythese techniques include formation of a slurry from one or moresolutions, which may contain water and one or more of the following:carbohydrates, proteins, lipids, stabilizers, vitamins and minerals. TheHMB is typically added to the carbohydrate slurry prior to the otherminerals. The slurry is emulsified, homogenized and cooled. Variousother solutions may be added to the slurry before processing, afterprocessing or at both times. The processed formula is then sterilizedand may be diluted to be dried to a powder, utilized on a ready-to-feedbasis or packaged in a concentrated liquid form. When the resultingformula is meant to be a ready-to-feed liquid or concentrated liquid, anappropriate amount of water would be added before sterilization.

Solid compositions such as bars, cookies, etc. may also be manufacturedutilizing techniques known to those skilled in the art. For example,they may be manufactured using cold extrusion technology as is known inthe art. To prepare such compositions, typically all of the powderedcomponents will be dry blended together. Such constituents typicallyinclude the proteins, vitamin premixes, certain carbohydrates, etc. Thefat-soluble components are then blended together and mixed with thepowdered premix above. Finally any liquid components are then mixed intothe composition, forming a plastic like composition or dough.

The process above is intended to give a plastic mass that can then beshaped, without further physical or chemical changes occurring, by theprocedure known as cold forming or extrusion. In this process, theplastic mass is forced at relatively low pressure through a die, whichconfers the desired shape. The resultant exudate is then cut off at anappropriate position to give products of the desired weight. If desiredthe solid product is then coated, to enhance palatability, and packagedfor distribution. Typically the package will provide directions for useby the end consumer (i.e., to be consumed by a cancer patient, to helpprevent lean muscle loss, etc.)

The solid compositions of the instant invention may also be manufacturedthrough a baked application or heated extrusion to produce cereals,cookies, and crackers. One knowledgeable in the arts would be able toselect one of the many manufacturing processes available to produce thedesired final product.

As noted above, the HMB may also be incorporated into juices,non-carbonated beverages, carbonated beverages, electrolyte solutions,flavored waters (hereinafter collectively “beverage”), etc. The HMB willtypically comprise from 0.5 to 2 gm/serving of the beverages. Methodsfor producing such beverages are well known in the art. The reader'sattention is directed to U.S. Pat. Nos. 6,176,980 and 5,792,502, thecontents of each which are hereby incorporated by reference. Forexample, all of the ingredients, including the HMB are dissolved in anappropriate volume of water. Flavors, colors, vitamins, etc. are thenoptionally added. The mixture is then pasteurized, packaged and storeduntil shipment.

METHODS OF USING THE INVENTION

It has now been surprisingly and unexpectedly discovered that HMB alonecan reduce tumor growth rate and in combination with sub-optimal doselevels of EPA enhance the anticachectic effect. The combination of EPAand HMB preserve muscle mass by attenuating protein degradation throughdown regulation of the increased expression of key regulatory componentsof the ubiquitin-proteasome proteolytic pathway. While not intending theinvention to be limited to any particular theory of operation,applicants describe below a probable mechanism.

In times of extreme need (e.g., starvation and the like), skeletalmuscle is often used by the body as a reservoir of amino acids andenergy. This is mediated by upregulation of the proteolysis anddownregulation of protein synthesis in muscle. The net result of whichis release of amino acids from muscle to the general circulation for usein maintenance of critical systems. When good health and adequatenutrient availability are restored, muscle is rebuilt. In the case ofcachexia, this system is inappropriately activated, so even in the caseof nutritional adequacy, muscle tissue proteins continue to be brokendown.

One of the key proteolytic systems which are inappropriately activatedis the ubiquitin proteosome system. When normally functioning, thissystem recognizes proteins which are either aged or in some other mannereither damaged or no longer needed, and marks them for removal viaconjugation with ubiquitin. Such ubiquitinylated proteins are recognizedby the proteosome, and degraded, releasing free ubiquitin and peptidesand free amino acids in an energy-consuming process. There are a numberof signaling molecules which activate or upregulate this system,including proteolysis-inducing factor (PIF), which is a protein factorproduced by certain cachexia-inducing tumors. Binding of PIF to themuscle cell causes the upregulation of phospholipase A (PLA). This inturn produces signaling factors which ultimately activate protein kinaseC, resulting in the activation of genes (via nuclear factor kappa B,NFκB) for ubiquitin conjugation and for certain subunits of theproteosome. The net result of all of this signaling is the up regulationof the ubiquitin proteosome system, and inappropriate, sustained proteindegradation in the muscle. FIG. 1 shows a detailed pathway of thisactivation sequence.

Protein Kinase C

Protein kinase C is a family of calcium- and lipid-activatedserine-threonine kinases that play a key role in numerous intracellularsignaling cascades. There are at least 12 different PKC isotypes, whichare grouped into three classes based on their primary structure andbiochemical properties (CA Carter: “Protein kinase C as a drug target:Implications for drug or diet prevention and treatment of cancer.”Current Drug Targets 1:163-183 (2000). These are theconventional—(cPKCα, βI, βII and γ) which require diacylglycerol,phosphatidylserine and calcium for activation, novel (nPKCδ, ε, η, θ andμ) which require diacylglycerol and phosphatidylserine, but are calciumindependent, and the atypical (aPKC λ, τ and ζ) which are calcium anddiacylglycerol-independent.

PKC is synthesized as a membrane-bound proenzyme. Removal of thepro-sequence by proteolytic cleavage, and subsequent phosphorylationreleases a competent enzyme from the membrane to the cytosol. Subsequentinteraction with the peculiar sets of activators produces active enzyme.Thus, there are several levels of regulation possible, including controlof expression, control of proteolytic processing, control of initialphosphorylation events and finally, regulation of the cytosolic levelsof the various activators required for full activity.

Protein kinase C is involved in some of the signaling pathways leadingto mitogenesis and proliferation of cells, apoptosis, plateletactivation, remodelling of the actin cytoskeleton, modulation of ionchannels and secretion. In addition, other observation that PKC is alsothe major receptor for tumor-promoting phorboly esters provided a keyreagent for studying the mechanism of action of this enzyme. PKC,regulates pathways relevant to inflammation, cardiovascular, peripheralmicrovascular, CNS, oncology, immune and infectious disease states, andare considered as serious and important targets for drug development.

NFκB

Nuclear Factor κ B (NFκB) is a family of transcription factors found ina wide variety of mammalian cells. The mature molecule is a homo- orheterodimer, made from one or two of the following 5 gene products (RelA(p65), p50, RelB, c-Rel and p52)—the most common is a dimer of RelA andp50. Under non-activated conditions, NFκB is localized in the cytosol byassociation with an inhibitory protein IκBα. Upstream signaling involvesan IκB kinase, and phosphorylation of the bound IκBα results in itsrelease from NFκB, allowing the later to translocate to the nucleus, andactivate specific gene transcription. The phosphorylated IκBα isdegraded by the ubiquitin-proteosome pathway.

NFκB is widely recognized as a key regulatory molecule associated withinflammation. Thus, it plays a key role in both acute and chronicinflammatory diseases (A B Lentsch and P A Ward: “Activation andregulation of NFκB during acute inflammation.” Clin Chem Lab Med37(3):205-208 (1999)). It also plays a role in certain aspects of otherdiseases, such as cancer metastasis (V B Andela, A H Gordon, G Zotalis,R N Rosier, J J Goater, G D Lewis, E M Schwarz, J E Puzas and R JO'Keefe: “NFκB: A pivotal transcription factor in prostate cancermetastasis to bone.” Clinical Orthopaedics and Related Research415S:S75-S85 (2003)). This transcription factor is involved in thedevelopment of the diabetic syndrome (E. Ho and T M Bray: “Antioxidants,NFκB activation and diabetogenesis.” Proceedings of the Society forExperimental Biology and Medicine 222:205-213 (1999)) and in immunedevelopment and regulation (J Moscat, M T Diaz-Meco and P Rennert: “NFκBactivation by proptein kinase C isoforms and B-cell function.” EMBOReports 4:31-36 (2003)). Finally, NFκB is associated with control ofapoptosis and in growth and differentiation. Indeed, PIF (proteolysisinducing factor, which is released by tumors and is involved incancer-induced lean mass losses) is thought to be a regulator ofembryonic development, and triggers a signaling cascade ultimatelythrough NFκB (F. Delfino and W H Walker: “Hormonal regulation of theNFκB signaling pathway.” Molecular and Cellular Endocrinology 157:1-9(1999); T M Watchorn, I Waddell, N Dowidar and J A Ross:“Proteolysis-inducing factor regulates hepatic gene expression via thetranscription factor NFκB and STST3.” FASEB J 15:562-564 (2001)).

It is also well known that EPA exerts it's beneficial effects oncachexia via inhibition of the signaling resulting from activation ofPLA, in particular the release of arachidonic acid (AA). This preventsthe subsequent upregulation and activation of the ubiquitin-proteosomepathway by removing the initial signaling event. HMB, while notpreventing the activation of PLA or the release of AA, does prevent theupregulation of protein kinase C, preventing all subsequent activationin the signaling pathway, also ultimately preventing the activation ofthe ubiquitin-proteosome system.

Any disease with which wasting or inflammation is associated such ascardiovascular, peripheral microvascular, central nervous system,oncology, immune and infectious disease states can be treated inaccordance with the present methods. Preferably, the disease is selectedfrom the group consisting of cancer, cachexia, age-associated wasting,wasting associated with long-term hospital stay, HIV/AIDS, arthritis,trauma, liver disease, Crohn's disease or other inflammatory boweldiseases (IBD), renal insufficiency and COPD (chronic obstructivepulmonary disease). More preferably, the disease is cachexia.

The present invention provides, in another embodiment, a method for thetreatment of the disease-associated wasting of a patient, such as amammal, preferably a human. The method comprises administering to thepatient the above-described composition, which comprises HMB in amountssufficient to treat the disease-associated wasting, wherein, uponadministration of the composition to the patient, the disease-associatedwasting is treated.

The amount of HMB that is sufficient to treat disease-associated wastingin a given patient can be determined in accordance with methods wellknown in the art. When treating the disease-associated wasting of apatient, desirably, the composition comprising HMB is administered to apatient suffering from disease-associated wasting in such an amount, insuch a manner, and over such a period of time that the patient's leantissue mass will increase without a concomitant decrease in thepatient's fat mass. An example, within the context of treating thecancer cachexia associated wasting of a human, when the composition isorally administered about twice a day for a minimum of two weeks; thedose is sufficient to provide at least about 2 gm HMB/day; for examplebetween 1 and 10 grams per day for a typical 70 kg person, more ideallybetween about 2 and 5 grams per day. The dosing on a body weight basismay range from about 0.01 to about 0.10 grams per kg body weight, moreideally between 0.02 and 0.07 grams/kg body weight.

Dosing for ω-3 fatty acids and EPA in particular are given above.

The present invention provides, in another embodiment, a method forreducing tumor growth rate in a patient, such as a mammal, preferably ahuman. The method comprises administering to the patient theabove-described composition, which comprises HMB in amounts sufficientto reduce tumor growth rate, wherein, upon administration of thecomposition to the patient, the tumor growth rate is reduced.

The amount of HMB that is sufficient to attenuate tumor growth in agiven patient can be determined in accordance with methods well known inthe art. When treating tumor growth in a patient, desirably, thecomposition comprising HMB is administered to a patient suffering fromtumor growth in such an amount, in such a manner, and over such a periodof time that the patient's tumor growth rate will decrease. An example,within the context of treating the tumor growth in an adult human, whenthe composition is orally administered about twice a day for a minimumof two weeks; the dose is sufficient to provide at least about 2 gmHMB/day.

The present invention provides, in another embodiment, a method for downregulating the expression and/or activity of protein kinase C. ExamplesI-IV show that both EPA and HMB attenuated PIF-induced activation ofprotein kinase C(PKC) and the subsequent degradation of IκBα and nuclearaccumulation of nuclear factor-κB (NF-κB).

The present invention provides, in another embodiment, a method for downregulating the expression and/or activity of nuclear factor kappa-B.Examples I-IV show that both EPA and HMB attenuated PIF-inducedactivation of protein kinase C(PKC) and the subsequent degradation ofIκBα and nuclear accumulation of nuclear factor-κB (NF-κB).

The present invention provides, in another embodiment, a method for downregulating the expression and/or activity of ubiquitin-conjugatingenzymes. Examples I-IV show that this was accompanied by a reduction inthe expression of E2_(14k) ubiquitin-conjugating enzyme. The combinationof EPA and HMB was at least as effective or more effective than eithertreatment alone. These results show that both EPA and HMB preservemuscle mass by attenuating protein degradation through down regulationof the increased expression of key regulatory components of theubiquitin-proteasome proteolytic pathway.

The present invention provides, in another embodiment, a method for downregulating the expression and/or activity of components of 26Sproteasome. Examples I-IV show that proteasome activity, determined bythe ‘chymotrypsin-like’ enzyme activity, was attenuated by HMB. Proteinexpression of the 20S α or β-subunits was reduced by at least 50%, aswere the ATPase subunits MSS1 and p42 of the 19S proteasome regulatorysubunit.

In addition to the above-mentioned methods of inhibiting proteindegradation, the present invention also provides, in another embodiment,a method for stimulating new protein synthesis in a patient having adisease-associated wasting condition, comprising administering aneffective amount of HMB. In such patients, the level of proteinsynthesis is usually depressed from normal as an effect of thedisease-associated conditions. In one aspect, protein synthesis in suchpatients is stimulated by at least 6%, 10%, 25%, 50% or more relative tothe pre-administration depressed levels. The status of muscle isdynamic, a net effect of both protein synthesis and degradation. It isunexpectedly and surprisingly found that HMB affects both processes inopposite directions—while protein synthesis is stimulated, proteindegradation is inhibited—such that the overall net gain in lean musclemass is magnified in wasting patients. The ratio of synthesis todegradation can thus be magnified 10 fold, 14 fold, 20 fold, 30 fold, 40fold or more. This is an ideal outcome for those suffering fromdisease-associated wasting conditions like cachexia, COPD and AIDS. Inanother aspect the stimulation is effected by an oral route ofadministration of the HMB. The HMB may be administered in combinationwith EPA; and may be administered with or without supporting protein,amino acids or other nutritional components.

Example I Prevention of Weight Loss and Attenuation of ProteinDegradation in Animals with Cancer Cachexia

This study evaluates the effect of HMB, in comparison with EPA orcombination, on weight loss induced by the MAC16 tumor and themechanisms involved. Weight loss induced by the MAC16 tumor is primarilyinduced by PIF.

Pure strain male NMRI mice (average weight 25 g) were obtained from ourown inbred colony and were transplanted with fragments of the MAC16tumor s.c. into the flank by means of a trochar, selecting from donoranimals with established weight loss as described in Bibby, M. C. et al.Characterization of a transplantable adenocarcinoma of the mouse colonproducing cachexia in recipient animals. J. Natl. Cancer Inst., 78:539-546, 1987. Transplanted animals were fed a rat and mouse breedingdiet (Special Diet Services, Witham, United Kingdom) and water adlibitum, and weight loss was evident 10-12 days after tumorimplantation. Animals just prior to the development of weight loss wererandomized to receive daily either EPA (in olive oil), HMB (in PBS) orthe combination as described in the figure legends administered p.o. bygavage, while control animals received either olive oil or PBS. EPA (98%as free acid) was purchased from Biomol Research Laboratories Inc., PA,USA. HMB (as the calcium salt) was obtained from Abbott Laboratories,Columbus, Ohio, USA. All groups contained a minimum of 6 mice. Tumorvolume, body weight and food and water intake were monitored daily.Animals were terminated by cervical dislocation when the body weightloss reached 25%, and all studies were conducted according to the UKCCRGuidelines for the care and use of laboratory animals. The soleusmuscles were quickly dissected out, together with intact tendons, andmaintained in isotonic ice-cold saline before determination of proteindegradation.

Freshly dissected soleus muscles were fixed via the tendons to aluminiumwire supports, under tension, at approximately resting length to preventmuscle shortening and preincubated for 45 min in 3 ml of oxygenated (95%oxygen:5% carbon dioxide) Krebs-Henseleit bicarbonate buffer (pH 7.4)containing 5 mM glucose and 0.5 mM cycloheximide. Protein degradationwas determined by the release of tyrosine over a 2 h period as describedin Waalkes, T. P. et al. A fluorimetric method for the estimation oftyrosine in plasma and tissues. J. Lab. Clin. Med., 50: 733-736, 1957.

Functional proteasome activity was determined by measuring the‘chymotrypsin-like’ enzyme activity, the predominant proteolyticactivity of the β-subunits of the proteasome according to the method ofOrino, E. et al. ATP-dependent reversible association of proteasomeswith multiple protein components to form 26S complexes that degradeubiquitinated proteins in human HL-60 cells. FEBS Lett., 284: 206-210,1991. Muscles were rinsed with ice-cold PBS, minced and sonicated in 20mM Tris. HCl, pH 7.5, 2 mM ATP, 5 mM MgCl₂ and 1 mM DTT. The sonicatewas then centrifuged for 10 min at 18,000 g, at 4° C. and thesupernatant was used to determine ‘chymotrypsin-like’ enzyme activity bythe release of aminomethyl coumarin (AMC) from the fluorogenic substratesuccinyl-LLVY-AMC. Activity was measured in the absence and presence ofthe specific proteasome inhibitor lactacystin (10 μM). Only lactacystinsuppressible activity was considered to be proteasome specific.

For Western blotting samples of soleus muscle cytosolic protein (2 to 5μg), obtained from the above assay, were resolved on 10% SDS-PAGE andtransferred to 0.45 μm nitrocellulose membrane (Hybond™, Amersham LifeScience Products, Bucks, United Kingdom), which had been blocked with 5%Marvel in PBS. The primary antibodies for MSS1 and p42 were used at adilution of 1:5000, for 20S proteasome α-subunits at 1:1500 and forβ-subunits at 1:1000, while the antibody for E2_(14k) was used at adilution of 1:500. The secondary antibodies were used at a dilution of1:2000. Mouse monoclonal antibodies to 20S proteasome subunits α 1, 2,3, 5, 6 and 7 (clone MCP 231), 20S proteasome subunit β3 (HC10), 19Sregulator ATPase subunit Rpt 1(S7, Mss1; clone MSS1-104) and 19Sregulator ATPase subunit Rpt 4 (S106, p42; clone p42-23) were purchasedfrom Affiniti Research Products, Exeter, United Kingdom. Rabbitpolyclonal antisera to ubiquitin-conjugating enzyme E2 (anti-UBC2antibody) was a gift from Dr. Simon Wing, McGill University, Montreal,Quebec, Canada. Peroxidase-conjugated goat anti-rabbit and rabbitanti-mouse secondary antibodies were from Dako Ltd., Cambridge, UnitedKingdom. Incubation was carried out for 2 h at room temperature, anddeveloped by chemiluminescence (ECL; Amersham).

A dose-response relationship of HMB on weight loss in mice bearing theMAC16 tumor is shown in FIG. 2. Doses of HMB greater than 0.125 g/kgcaused a significant reduction in weight loss (FIG. 2A). Differencesfrom the control group are indicated as a, p<0.05; b, p<0.01 and c,p<0.005. Attenuation of weight loss was not accompanied by an alterationin food and water intake. A dose level of 0.25 g/kg was chosen for allsubsequent experiments. The effect of HMB, EPA and the combination ofHMB and EPA on weight loss in MAC16 cachectic tumour-bearing mice isshown in FIG. 3. Differences from the control group are indicated as a,p<0.05; b, p<0.01 or c, p<0.005. A suboptimal dose of EPA was chosen toinvestigate interactions with HMB. All treatments caused a significantincrease in soleus muscle weight (FIG. 4A), and a significant reductionin tyrosine release (FIG. 4B), indicating a reduction in total proteindegradation. Differences from the PBS control group are indicated as a,p<0.05, b, p<0.01 or c, p<0.005. At the doses chosen, HMB was aseffective as EPA.

Proteasome expression has been shown to be elevated in gastrocnemiusmuscles of mice bearing the MAC16 tumor and this increased geneexpression has been shown to be attenuated by EPA. The results in FIG. 5show that functional proteasome activity, as determined by‘chymotrypsin-like’ enzyme activity, was attenuated by HMB to the sameextent as EPA at the doses chosen, and that the combination of HMB andEPA did not produce a further depression in activity. Differences fromcontrol are indicated as c, p<0.005. Protein expression of proteasomesubunits was analysed by Western blotting of supernatants from sonicatedmuscle tissues. Expression of 20S proteasome α-subunits, the structuralunits of the proteasome was attenuated by both HMB and EPA, and therewas some indication of a further decrease of band 2 for the combination(FIG. 6A). Differences from control are shown as c, p<0.001, whiledifferences from HMB are shown as e, p<0.01. Expression of the 20Sproteasome β-subunits, the catalytic subunits of the proteasome, werealso attenuated by HMB and EPA, but the combination was more effectivethan either agent alone (FIG. 6B). Differences from control are shown asc, p<0.001.

Expression of MSS1, an ATPase subunit of the 19S proteasome regulatorycomplex is shown in FIG. 7A. Both HMB and EPA attenuated MSS1expression, but the combination did not appear to produce a furtherreduction. Similar results were obtained with p42, another ATPasesubunit of the 19S regulator, that promotes ATP dependent association ofthe 20S proteasome with the 19S regulator to form the 26S proteasome(FIG. 7B). Differences from control are shown as c, p<0.001. Again bothHMB and EPA appeared to be equally effective, while the combination didappear to reduce p42 expression further. Expression of theubiquitin-conjugating enzyme, E2_(14k), was also reduced by both HMB andEPA, while the combination caused a further reduction in expression(FIG. 8). Differences from control are shown as b, p<0.01 and c,p<0.001, while differences from HMB alone are shown as d, p<0.05 and f,p<0.001. These results confirm HMB to be as effective as EPA inattenuating loss of muscle mass, protein degradation and down-regulationof the ubiquitin-proteasome proteolytic pathway, and this mechanismappears to be responsible for the preservation of muscle mass incachectic mice bearing the MAC16 tumor.

This study has shown that HMB is effective in attenuating thedevelopment of cachexia or involuntary weight loss in mice bearing theMAC16 tumor and produced a reduction in protein degradation in skeletalmuscle by down regulating the increased expression of theubiquitin-proteasome pathway. Thus HMB is as effective as EPA inreducing protein expression of the 20S proteasome α0 and β subunits, aswell as two subunits of the 19S regulator MSS1 and p42, expression ofE2_(14k) and proteasome proteolytic activity.

Example II Attenuation of Tumor Growth in Animals

The animal study described in Example I above also evaluated the effectof HMB on tumor growth rate in MAC16 cachectic tumor-bearing mice. Theexperiment was conducted as described in Example I.

A dose-response relationship of HMB alone on tumor growth rate in micebearing the MAC16 tumor is shown in FIG. 2B. Differences from thecontrol group are indicated as a, p<0.05; b, p<0.01 and c, p<0.005.Doses of HMB greater than 0.125 g/kg caused a significant reduction intumor growth rate. Attenuation of tumor growth was not accompanied by analteration in food and water intake.

Example II Attenuation of Protein Degradation in Murine Myotubes

This study examines the effect of HMB on PIF-induced protein degradationand signalling pathways in murine myotubes to determine the mechanism ofthe attenuation of the increased expression of the ubiquitin-proteasomeproteolytic pathway.

C₂C₁₂ myotubes were routinely passaged in DMEM supplemented with 10%FCS, glutamine and 1% penicillin-streptomycin under an atmosphere of 10%CO₂ in air at 37□C. Myotubes were formed by allowing confluent culturesto differentiate in DMEM containing 2% HS, with medium changes every 2days.

PIF was purified from solid MAC16 tumors (Todorov, P. et al.Characterization of a cancer cachectic factor. Nature, 379: 739-742,1996.) excised from mice with a weight loss of 20 to 25%. Tumors werehomogenised in 10 mM Tris-HCl, pH 8.0, containing 0.5 mMphenylmethylsulfonyl fluoride, 0.5 mM EGTA and 1 mM dithiothreitol at aconcentration of 5 ml/g tumor: Solid ammonium sulfate was added to 40%w/v and the supernatant, after removal of the ammonium sulfate, wassubjected to affinity chromatography using anti-PIF monoclonal antibodycoupled to a solid matrix as described in Todorov, P. et al Induction ofmuscle protein degradation and weight loss by a tumor product. CancerRes., 56: 1256-1261, 1996. The immunogenic fractions were concentratedand used for further studies.

Myotubes in six-well multidishes were labeled with L-[2, 6⁻³H]phenylalanine (0.67 mCi/mmole) for 24 h in 2 ml DMEM containing 2% HS.They were then washed three times in PBS followed by a 2 h incubation at37° C. in DMEM without phenol red until no more radioactivity appearedin the supernatant. These myotubes were then further incubated for 24 hin the presence of PIF, with and without EPA or HMB, in fresh DMEMwithout phenol red, to prevent quenching of counts, and in the presenceof 2 mM cold phenylalanine to prevent reincorporation of radioactivity.The amount of radioactivity released into the medium was expressed as apercentage of control cultures not exposed to PIF to determine totalprotein degradation.

For measurement of arachidonic acid release, myotubes in six-well multidishes containing 2 ml DMEM with 2% HS were labeled for 24 h with 10 μMarachidonic acid (containing 1 μCi of [³H] arachidonate/ml) (Smith, H.et al. Effect of a cancer cachectic factor on proteinsynthesis/degradation in murine C₂C₁₂ myoblasts: Modulation byeicosapentaenoic acid. Cancer Res., 59: 5507-5513, 1999). Cells werethen washed extensively with PBS to remove traces of unincorporated [³H]arachidonate and either EPA or HMB was added 2 h prior to PIF. After afurther 24 h 1 ml of medium was removed to determine the radioactivityreleased.

The functional activity of the β subunits of the proteasome wasdetermined as the ‘chymotrypsin-like’ enzyme activity obtainedfluorimetrically according to the method of Orino, E. et al.ATP-dependent reversible association of proteasomes with multipleprotein components to form 26S complexes that degrade ubiquitinatedproteins in human HL-60 cells. FEBS Lett., 284: 206-210, 1991. Myotubeswere exposed to PIF for 24 h with or without EPA or HMB added 2 h priorto PIF and enzyme activity was determined in a supernatant fraction(Whitehouse, A. S. et al. Increased expression of theubiquitin-proteasome pathway in murine myotubes by proteolysis-inducingfactor (PIF) is associated with activation of the transcription factorNF-κB. Br. J. Cancer, 89: 1116-1122, 2003) by the release of aminomethylcoumarin (AMC) from succinyl-LLVY-AMC (0.1 mM) in the presence orabsence of the specific proteasome inhibitor lactacystin (10 μM)(Fenteany, G. et al. Lactacystin, proteasome function and cell fate. J.Biol. Chem., 273: 8545-8548, 1998). Only lactacystin suppressibleactivity was considered to be proteasome specific. Activity was adjustedfor the protein concentration of the sample, determined using theBradford assay (Sigma Chemical Co., Dorset, United Kingdom) using bovineserum albumin as standard.

For Western blot analysis, cytosolic protein (2 to 5 μg) obtained forthe above assay were resolved on 10% SDS-PAGE and transferred to 0.45 μmnitrocellulose membrane, which had been blocked with 5% Marvel in PBS,at 4° C. overnight. The primary antibodies were used at a dilution of1:100 (anti-actin and PKC_(α)); 1:500 (anti-ERK1 and 2); 1:1000(anti-20S proteasome β-subunit and I-κBα); 1:1500 (anti-20S proteasomeα-subunit) or 1:5000 (anti-p42), while the secondary antibodies wereused at a dilution of 1:2000. Incubation was carried out for 2 h at roomtemperature and development was by ECL. Loading was quantitated by actinconcentration.

DNA binding proteins were extracted from myotubes by the method ofAndrews, N. C. et al. A rapid micropreparation technique for extractionof DNA-binding proteins from limiting numbers of mammalian cells.Nucleic Acids Res., 19: 2499, 1991, which utilizes hypotonic lysisfollowed by high salt extraction of nuclei. The EMSA (electrophoreticmobility shift assay) binding assay was carried out according to themanufacturer's instructions.

Since protein degradation and activation of the ubiquitin-proteasomeproteolytic pathway in mice bearing the MAC16 tumor is thought to bemediated by PIF, mechanistic studies on the effect of HMB on proteindegradation were carried out in murine myotubes treated with PIF.PIF-induced total protein breakdown with a typical bell-shapeddose-response curve, as previously reported by Gomes-Marcondes, et alDevelopment of an in-vitro model system to investigate the mechanism ofmuscle protein catabolism induced by proteolysis-inducing factor. Br. J.Cancer, 86: 1628-1633, 2002 with a maximal effect at 4 nM. The effect ofEPA has been previously shown (Smith, H. J. et al. Effect of a cancercachectic factor on protein synthesis/degradation in murine C₂C₁₂myoblasts: Modulation by eicosapentaenoic acid. Cancer Res., 59:5507-5513, 1999; Whitehouse, A. S. et al. Induction of proteincatabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid throughincreased expression of the ubiquitin-proteasome pathway. Br. J. Cancer,89: 737-745, 2003; and Whitehouse, A. S. et al. Increased expression ofthe ubiquitin-proteasome pathway in murine myotubes byproteolysis-inducing factor (PIF) is associated with activation of thetranscription factor NF-κB. Br. J. Cancer, 89: 1116-1122, 2003) to beeffective at 50 μM, and the data in FIG. 9A shows that at aconcentration of 50 μM both HMB and EPA were equally effective inattenuating PIF induced protein degradation. There was also someattenuation at 25 μM HMB at low, but not at high concentrations of PIF.Differences from control in the absence of PIF are indicated as a,p<0.005, while differences form control with PIF (for groups withadditions of HMV or EPA) are indicated as b, p<0.01 and c, p<0.005.

PIF-induced protein degradation has previously been shown to be due toan increased expression of the regulatory components of theubiquitin-proteasome proteolytic pathway by Lorite, M. J., Smith, H. J.,Arnold, J. A., Morris, A., Thompson, M. G. and Tisdale, M. J. Activationof ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo andmurine myoblasts in vitro by a proteolysis-inducing factor (PIF). Br. J.Cancer, 85: 297-302, 2001 and Gomes-Marcondes, M. C. C., Smith, H. J.,Cooper, J. C. and Tisdale, M. J. Development of an in-vitro model systemto investigate the mechanism of muscle protein catabolism induced byproteolysis-inducing factor. Br. J. Cancer, 86: 1628-1633, 2002.

The functional activity of this pathway is measured by the‘chymotrypsin-like’ enzyme activity, the predominant proteolyticactivity of the β-subunits of the proteasome. PIF induced an increase in‘chymotrypsin-like’ enzyme activity, which was maximal at 4.2 nM. Theeffect of PIF was completely attenuated by 50 μM EPA and both 25 and 50μM HMB. (FIG. 9B, differences from control are shown as a, p<0.001,while differences in the presence of EPA or HMB are shown as b,p<0.001). A similar effect was observed on expression of proteasome 20Sα subunits, β subunits and p42, an ATPase subunit of the 19S regulatorthat promotes ATP-dependent association of the 20S proteasome with the19S regulator to form the 26S proteasome (FIG. 10). In all casesexpression was increased by PIF at 4.2 and 10 nM and this was attenuatedby EPA and HMB at 50 μM, but not at 25 μM. These results confirm thatHMB attenuates protein degradation through an effect on PIF induction ofthe ubiquitin-proteasome pathway.

Example IV Effect on Activity of Mediator of Signaling in Inflammationand Proteolysis

The in vitro study described in Example III above also evaluated theeffect of HMB on molecules that are key mediators in the pathway ofinflammation. This experiment was conducted as described in Example III.

Activation of PKC has been shown to activate extracellularsignal-regulated kinase (ERK) cascade of MAPK signalling pathways(Toker, A. Signalling through protein kinase C. Front. Biosci., 3:1134-1147, 1998; Wolf, I. and Seger, R. The mitogen-activated proteinkinase signalling cascade: from bench to bedside. IMAJ., 4: 641-647).The activated ERKs, e.g., ERK1 (or p44 MAPK) and ERK2 (or p42 MAPK), areable to phosphorylate and consequently activate cytosolic phospholipaseA2, the rate-limiting enzyme in pathways involving arachidonic acidrelease in inflammation. In addition, PIF has been shown to inducephosphorylation of p42/44 MAPK, while the total MAPK remained unchangedand to be involved in PIF-induced proteasome expression (Smith, H. J. etal. Signal transduction pathways involved in proteolysis-inducing factorinduced proteasome expression in murine myotubes. Br. J. Cancer, 89:1783-1788, 2003). The effect of EPA and HMB on this process is shown inFIG. 12. PIF induced an increased phosphorylation of p42/44 that wasmaximal at 4.2 nM and this effect was completely attenuated by both EPAand HMB at 50 μM, but not HMB at 25 μM. The ability of HMB to attenuateERK ½ phosphorylation may be important in inhibition of PIF-inducedproteasome expression by HMB.

Experiments using mutants of PKC as well as inhibitors of this enzymeshow that this forms a central mediator of intracellular signalling byPIF. PKC is likely to be involved in phosphorylation (and degradation)of IκBα leading to nuclear accumulation of NF-κB and increased genetranscription. PIF stimulates translocation of PKC_(α) from thecytoplasm to the plasma membrane (FIG. 11) resulting in activation witha maximum effect at 4.2 nM PIF as with protein degradation (FIG. 9).This process was effectively attenuated by both EPA and HMB at 50 μM;while HMB was less effective at 25 μM (FIG. 11). This suggests thatPIF-induced stimulation of PKC is attenuated by HMB through inhibitionof PKC.

As previously discussed PIF induces degradation of IκBα and stimulatesnuclear accumulation of NF-κB and this process has been shown to beattenuated by 50 μM EPA (Whitehouse, A. S. et al. Increased expressionof the ubiquitin-proteasome pathway in murine myotubes byproteolysis-inducing factor (PIF) is associated with activation of thetranscription factor NF-κB. Br. J. Cancer, 89: 1116-1122, 2003). Theresults in FIG. 13A show HMB at 50 μM to effectively attenuate IκBαdegradation in the presence of PIF in murine myotubes, and preventnuclear accumulation of NF-κB (FIG. 13C). Differences from 0 nM PIF areshown as b, p<0.01 and c, p<0.001. Only partial inhibition of binding ofNF-κB to DNA was observed when HMB was used at a concentration of 25 μM(FIG. 13B). Differences from 0 nM PIF b=p<0.01 and c=p<0.001.Differences between 50 uM HMB and PIF treated against PIF alone at thesame concentration e=p<0.01 and f=p<0.001. These results suggest thatthe overall effect of HMB is comparable to that of EPA in preventingmovement of NF-κB into the nucleus with concomitant activation of geneexpression.

Thus HMB appears to be an effective agent in the treatment of cytokineinduced inflammation and muscle wasting in cancer cachexia. HMB appearsto exert its effect by inhibition of PKC activity, and resultantstabilization of the cytoplasmic IκB/NF-κB complex. Since thesemolecules are key mediators in the pathway of inflammation, HMB appearsto be an anti-inflammatory compound.

Example V Composition of a Nutritional Product to Prevent InvoluntaryWeight Loss

The specific list of materials for manufacturing the nutritional productof this Example is presented in Table 1. Of course, various changes inspecific ingredients and quantities may be made without departing fromthe scope of the invention.

TABLE 1 LIST OF MATERIALS AMOUNT INGREDIENT (KG) WATER 316ULTRATRACE/TRACE MINERAL PREMIX 0.06 ZINC SULFATE 0.033 MANGANESESULFATE 0.0082 SODIUM MOLYBDATE 0.00023 CHROMIUM CHLORIDE 0.00029 SODIUMSELENITE 0.000098 POTASSIUM CHLORIDE 0.072 SODIUM CITRATE 2.89 POTASSIUMIODIDE 0.00009 POTASSIUM CITRATE 1.5 CORN SYRUP 7.68 MALTODEXTRIN 53.6MAGNESIUM PHOSPHATE DIBASIC 0.26 CALCIUM PHOSPHATE TRIBASIC 0.99MAGNESIUM CHLORIDE 1.2 SUCROSE 11.9 FRUCTOOLIGOSACCHARIDE 5.9 MEDIUMCHAIN TRIGLYCERIDE 2.6 CANOLA OIL 1.5 SOY OIL 0.87 57% VITAMIN APALMITATE 0.007 VITAMIN DEK PREMIX 0.04 VITAMIN D 0.0000088D-ALPHA-TOCOPHEROL ACETATE 0.036 PHYLLOQUINONE 0.00006 CARRAGEENAN 0.03SOY LECITHIN 0.6 SODIUM CASEINATE 15.5 CALCIUM CASEINATE 4.2 CALCIUM HMBMONOHYDRATE 2.6 MILK PROTEIN ISOLATE 14 REFINED DEODORIZED SARDINE OIL6.9 ASCORBIC ACID 0.12 45% POTASSIUM HYDROXIDE 0.13 TAURINE 0.12 WATERSOLUBLE VITAMIN PREMIX 0.11 NIACINAMIDE 0.017 CALCIUM PANTOTHENATE 0.01THIAMINE CHLORIDE HYDROCHLORIDE 0.003 PYRIDOXINE HYDROCHLORIDE 0.003RIBOFLAVIN 0.002 FOLIC ACID 0.0004 BIOTIN 0.00034 CYANOCOBALAMIN0.000038 ASCORBYL PALMITATE 0.03 CHOLINE CHLORIDE 0.25 L-CARNITINE0.0681 N&A MARSHMALLOW VANILLA 1.6 N&A DULCE DE LECHE 0.27

The liquid nutritional product of the present invention was manufacturedby preparing three slurries which are blended together, combined withrefined deodorized sardine oil, heat treated, standardized, packaged andsterilized. The process for manufacturing 454 kg (1,000 pounds) of theliquid nutritional product, using the List of Materials from Table 7, isdescribed in detail below.

A carbohydrate/mineral slurry is prepared by first heating about 62.6 kgof water to a temperature in the range of about 71° C. to 77° C. withagitation. The HMB is added to the water and dissolved by agitating theresultant solution for at least five minutes. The required amount ofpotassium citrate and ultratrace/trace mineral premix is added to thewater and dissolved by agitating the resultant solution for at least 10minutes. The following minerals are then added, in the order listed,with high agitation: magnesium chloride, potassium chloride, sodiumcitrate, potassium iodide, magnesium phosphate and tricalcium phosphate.The slurry is allowed to mix under moderate agitation until completelydissolved or dispersed. The corn syrup, sucrose and maltodextrin arethen added to the slurry with agitation. Add the required amount of FOSand allow to mix. The completed carbohydrate/mineral slurry is held withhigh agitation at a temperature in the range of about 60-66° C. for notlonger than 8 hours until it is blended with the other slurries.

An oil slurry is prepared by combining and heating the medium chaintriglycerides (fractionated coconut oil), canola oil and soy oil to atemperature in the range of about 32-43° C. with agitation. The vitaminDEK premis is added and allowed to mix until completely dispersed. Therequired amounts of following ingredients are added: sly lecithin,vitamin A, ascorbyl plamitate, and vitamin E. The carrageen is added andallowed to mix until completely dispersed. The completed oil slurry isheld under moderate agitation at a temperature in the range of about32-43° C. for not longer than 8 hours until it is blended with the otherslurries.

A protein slurry is prepared by first heating about 196.78 kg of waterto a temperature in the range of about 60-63° C. with agitation. Thecalcium caseinate and sodium caseinate and milk protein isolate areblended into the slurry using a mixing apparatus. The completed proteinslurry is held under agitation at a temperature in the range of about54-60° C. for not longer than 2 hours before being blended with theother slurries.

The oil and the protein slurry are blended together with agitation andthe resultant blended slurry is maintained at a temperature in the rangeof about 54-66° C. After waiting for at least five minutes thecarbohydrate/mineral slurry is added to the blended slurry from thepreceding step with agitation and the resultant blended slurry ismaintained at a temperature in the range of about 54-66° C. The refineddeodorized sardine oil is then added to the slurry with agitation. (In amost preferred method of manufacture the sardine oil would be slowlymetered into the product as the blend passes through a conduit at aconstant rate.) Preferably after at least 5 minutes the pH of theblended slurry is determined. If the pH of the blended slurry is below6.55, it is adjusted with dilute potassium hydroxide to a pH of 6.5 to6.8.

After waiting a period of not less than one minute nor greater than twohours the blended slurry is subjected to deaeration,Ultra-High-Temperature (UHT) treatment, and homogenization, as describedas follows: use a positive pump for supplying the blended slurry forthis procedure; heat the blended slurry to a temperature in the range ofabout 66-71° C.; deaerate the blended slurry to 25.4-38.1 cm of Hg;emulsify the blended slurry at 61-75 Atmospheres; heat the blendedslurry to a temperature in the range of about 120-122° C. by passing itthrough a plate/coil heat exchanger with a hold time of approximately 10seconds; UHT heat the blended slurry to a temperature in the range ofabout 144-147° C. with a hold time of approximately 5 seconds; reducethe temperature of the blended slurry to be in the range of about120-122° C. by passing it through a flash cooler; reduce the temperatureof the blended slurry to be in the range of about 71-82° C. by passingit through a plate/coil heat exchanger; homogenize the blended slurry atabout 265 to 266 Atmospheres; pass the blended slurry through a holdtube for at least 16 seconds at a temperature in the range of about74-85° C.; and cool the blended slurry to a temperature in the range ofabout 1-70° C. by passing it through a large heat exchanger.

Store the blended slurry at a temperature in the range of about 1-7° C.,preferably with agitation.

Preferably at this time appropriate analytical testing for qualitycontrol is conducted. Based on the test results an appropriate amount ofdilution water (10-38° C.) is added to the blended slurry withagitation.

A vitamin solution and flavor solution are prepared separately and thenadded to the blended slurry.

The vitamin solution is prepared by heating about 3.94 kg of water to atemperature in the range of about 43-66° C. with agitation, andthereafter adding the following ingredients, in the order listed:Ascorbic Acid, 45% Potassium Hydroxide, Taurine, Water Soluble VitaminPremix, Choline Chloride, and L-Carnitine. The vitamin solution is thenadded to the blended slurry with agitation.

The flavor solution is prepared by adding the marshmallow and dulce deleche flavor to about 7.94 kg of water with agitation. A nutritionalproduct according to the present invention has been manufactured usingan artificial marshmallow flavor distributed by Firmenich Inc.,Princeton, N.J., U.S.A. and a natural & artificial dulce de leche flavordistributed by Firmenich Inc. The flavor solution is then added to theblended slurry with agitation.

If necessary, diluted potassium hydroxide is added to the blended slurrysuch that the product will have a pH in the range of 6.4 to 7.0 aftersterilization. The completed product is then placed in suitablecontainers and subjected to sterilization. Of course, if desired asepticprocessing could be employed.

Example VI Composition of a Nutritional Product to Control GlycemicResponse

Table 2 presents a bill of materials for manufacturing 1,000 kg of aliquid nutritional product, which provides nutrients to a person butlimits resulting insulin response. A detailed description of itsmanufacture follows.

TABLE 2 Bill of Materials for a Liquid Nutritional Ingredient Quantityper 1,000 kg Water QS Maltodextrin 56 kg Acidc casein 41.093 kg Fructose28 kg High oleic safflower oil 27.2 kg Maltitol syrup 16 kg Maltitol12.632 kg Fibersol ® 2(E) 8.421 kg Caseinate 6.043 kgFructooligosaccharide 4.607 kg Soy polysaccharide 4.3 kg Canola oil 3.2kg Tricalcium phosphate 2.8 kg Magnesium chloride 2.4 kg Lecithin 1.6 kgSodium citrate 1.18 kg Potassium citrate 1.146 kg Sodium hydroxide 1.134kg Magnesium phosphate 1.028 kg Calcium HMB monohydrate 5.7 kgm-inositol 914.5 gm Vitamin C 584 gm Potassium chloride 530 gm Cholinechloride 472.1 gm 45% Potassium hydroxide 402.5 gm UTM/TM premix 369.3gm Potassium phosphate 333 gm Carnitine 230.5 gm Gellan gum 125 gmTtaurine 100.1 gm Vitamin E 99 gm Lutein Esters (5%) 92 gm WSV premix75.4 gm Vitamin DEK premix 65.34 gm 30% Beta carotene 8.9 gm Vitamin A8.04 gm Pyridoxine hydrochloride 3.7 gm Chromium chloride 1.22 gm Folicacid 0.64 gm Potassium iodide 0.20 gm Cyanocobalamin 0.013 gm WSV premix(per g premix): 375 mg/g niacinamide, 242 mg/g calcium pantothenate, 8.4gm/g folic acid, 62 mg/g thiamine chloride hydrochloride, 48.4 gm/griboflavin, 59.6 mg/g pyridoxine hydrochloride, 165 mcg/g cyanocobalaminand 7305 mcg/g biotin Vitamin DEK premix (per g premix): 8130 IU/gvitamin D₃, 838 IU/g vitamin E, 1.42 mg/g vitamin K₁ UTM/TM premix (perg premix): 45.6 mg/g zinc, 54 mg/g iron, 15.7 manganese, 6.39 mg/gcopper, 222 mcg/g selenium, 301 mcg/g chromium and 480 mcg/g molybdenium

The diabetic liquid nutritional products of the present invention aremanufactured by preparing four slurries that are blended together, heattreated, standardized, packaged and sterilized.

A carbohydrate/mineral slurry is prepared by first heating about 82 kgof water to a temperature of from about 65° C. to about 71° C. withagitation. With agitation, the required mount of calcium HMB is addedand agitated for 5 minutes. The required amount of sodium citrate andgellen gum distributed by the Kelco, Division of Merck and CompanyIncorporated, San Diego, Calif., U.S.A. is added and agitated for 5minutes. The required amount of the ultra trace mineral/trace mineral(UTM/TM) premix (distributed by Fortitech, Schnectady, N.Y.) is added.The slurry is greenish yellow in color. Agitation is maintained untilthe minerals are completely dispersed. With agitation, the requiredamounts of the following minerals are then added: potassium citrate,potassium chloride, chromium chloride, magnesium chloride and potassiumiodide. Next, the first maltodextrin distributed by Grain ProcessingCorporation, Muscataine, Iowa, U.S.A. and fructose are added to slurryunder high agitation, and are allowed to dissolve. With agitation, therequired amounts of maltitol powder distributed by Roquette America,Inc., Keokuk, Iowa, maltitol syrup distributed by AlGroup Lonza, FairLawn, N.J., fructooligosaccharides distributed by Golden TechnologiesCompany, Golden, Colo., U.S.A. and a second maltodextrin distributed byMatsutani Chemical Industry Co., Hyogo, Japan under the product nameFibersol® 2(E) are added and agitated well until completely dissolved.The required amount of tricalcium phosphate and magnesium phosphate areadded to the slurry under agitation. The completed carbohydrate/mineralslurry is held with agitation at a temperature from about 65° C. toabout 71° C. for not longer than twelve hours until it is blended withthe other slurries.

A fiber in oil slurry is prepared by combining and heating the requiredamounts of high oleic safflower oil and canola oil to a temperature fromabout 40.5° C. to about 49° C. with agitation. With agitation, therequired amounts of lutein esters from Cognis of LaGrange, Ill. isadded. Agitate for a minimum of 15 minutes. With agitation, the requiredamounts of the following ingredients are added to the heated oil:lecithin (distributed by Central Soya Company, Fort Wayne, Ind.),Vitamin D, E, K premix (distributed by Vitamins Inc., Chicago, Ill.),vitamin A, vitamin E and beta-carotene. The required amounts of soypolysaccharide distributed by Protein Technology International, St.Louis, Mo. is slowly dispersed into the heated oil. The completedoil/fiber slurry is held under moderate agitation at a temperature fromabout 55° C. to about 65° C. for a period of no longer than twelve hoursuntil it is blended with the other slurries.

A first protein in water slurry is prepared by heating 293 kg of waterto 60° C. to 65° C. With agitation, the required amount of 20% potassiumcitrate solution is added and held for one minute. The required amountof acid casein is added under high agitation followed immediately by therequired amount of 20% sodium hydroxide. The agitation is maintained athigh until the casein is dissolved. The slurry is held from about 60° C.to 65° C. with moderate agitation.

A second protein in water slurry is prepared by first heating about 77kg of water to a temperature of about 40° C. with agitation. Thecaseinate is added and the slurry is agitated well until the caseinateis completely dispersed. With continued agitation, the slurry is slowlywarmed to 60° C. to 65° C. The slurry is held for no longer than twelvehours until it is blended with the other slurries.

The batch is assembled by blending 344 kg of protein slurry one with 84kg of protein slurry two. With agitation, the 37 kg of the oil/fiberslurry is added. After waiting for at least one minute, 216 kg of thecarbohydrate/mineral slurry is added to the blended slurry from thepreceding step with agitation and the resultant blended slurry ismaintained at a temperature from about 55° C. to about 60° C. The pH ofthe blended batch is adjusted to a pH of 6.45 to 6.75 with 1N potassiumhydroxide.

After waiting for a period of not less than one minute nor greater thantwo hours, the blend slurry is subjected to deaeration,ultra-high-temperature treatment, and homogenization. The blended slurryis heated to a temperature from about 71° C. to about 82° C. anddeaerated under vacuum. The heated slurry is then emulsified through asingle stage homogenizer at 900 to 1100 psig. After emulsification, theslurry is heated from about 99° C. to about 110° C. and then heated to atemperature of about 146° C. for about 5 seconds. The slurry is passedthrough a flash cooler to reduce the temperature to from about 99° C. toabout 110° C. and then through a plate cooler to reduce the temperatureto from about 71° C. to about 76° C. The slurry is then homogenized at3900 to 4100/400 to 600 psig. The slurry is held at about 74° C. toabout 80° C. for 16 seconds and then cooled to 1° C. to about 7° C. Atthis point, samples are taken for microbiological and analyticaltesting. The mixture is held under agitation.

A water soluble vitamin (WSV) solution is prepared separately and addedto the processed blended slurry.

The vitamin solution is prepared by adding the following ingredients to9.4 kg of water with agitation: WSV premix (distributed by J.B.Laboratories, Holland, Mich.), vitamin C, choline chloride, L-carnitine,taurine, inositiol, folic acid, pyridoxine hydrochloride andcyanocobalamin. The required amount of 45% potassium hydroxide slurry isadded to bring the pH to between 7 and 10.

Based on the analytical results of the quality control tests, anappropriate amount of water is added to the batch with agitation toachieve desired total solids. Additionally, 8.8 kg of vitamin solutionis added to the diluted batch under agitation. The product pH may beadjusted to achieve optimal product stability. The completed product isthen placed in suitable containers and subjected to terminalsterilization.

Example VII Composition of a Pediatric Nutritional Product

Table 3 presents a bill of materials for manufacturing 771 kg of apediatric enteral nutritional of the instant invention. A detaileddescription of its manufacture follows.

TABLE 3 Bill of materials for vanilla pediatric nutritional IngredientQuantity per 771 kg Stock PIF Slurry High oleic safflower oil 40.7 kgSoy oil 24.4 kg MCT oil 16.3 kg Lecithin 840.2 g Monoglycerides 840.2 gCarrageenan 508.9 g Caseinate 32.8 kg Stock OSV blend DEK premix 83.3 gVitamin A 7.1 g Lutein esters (5%) 92 g Stock PIW slurry Water 530 kgCaseinate 11.3 kg Whey protein 11.9 kg Stock MIN slurry Water 18 kgCellulose gum 1696 g Calcium HMB monohydrate 4.4 kg Magnesium chloride2.7 kg Potassium chloride 1.0 kg Potassium citrate 2.7 kg Potassiumiodide 0.25 g Dipotassium phosphate 1.45 kg Final blend PIW slurry 251kg PIF slurry 53 kg MIN slurry 12.6 kg Sodium chloride 127.4 g Sucrose77.6 kg Tricalcium phosphate 2.5 kg Water 167 kg Stock WSV solutionWater 31.7 kg Potassium citrate 3.74 g UTM/TM premix 172.2 g WSV premix134.1 g m-inositol 176.7 g Ttaurine 145.5 g L-carnitine 34.92 g Cholinechloride 638.7 g Stock ascorbic acid solution Water 18.6 kg Ascorbicacid 550.0 g 45% KOH 341 g Stock vanilla solution Water 38.5 kg Vanillaflavor 4.3 kg DEK premix: (per gm premix) 12,100 IU vitamin D₃, 523 IUvitamin E, 0.962 mg vitamin K₁ UTM/TM premix: (per gm premix) 132 mgzinc, 147 mg iron, 10.8 mg manganese, 12.5 mg copper, 0.328 mg selenium,0.284 mg molybdenum WSV premix: (per gm premix) 375 mg niacinamide, 242mg d-calcium pantothenate, 8.4 mg folic acid, 62 mg thiamine chloridehydrochloride, 48.4 mg riboflavin, 59.6 mg pyridoxine hydrochloride,165.5 mcg cyanocobalamin, 7305 mcg biotin

The stock oil soluble vitamin blend (OSV blend) is prepared by weighingout the specified amount of DEK premix into a screw cap, light protectedcontainer large enough to hold 54 g of oil soluble vitamins. Using aplastic pipette, the required amount of vitamin A is added to the DEKaliquot. The container is flushed with nitrogen prior to applying thelid.

The stock protein in fat slurry (PIF) was prepared by adding therequired amounts of high oleic safflower oil, soy oil and MCT oil to theblend tank. The mixture is heated to 40.5° C. to 49° C. with agitation.With agitation, the required amounts of lutein esters from AmericanRiver Nutrition of Hadley, Mass. is added. Agitate for a minimum of 15minutes. The emulsifiers, lecithin (distributed by Central Soya ofDecatur, Ind.) and monoglycerides (distributed by Quest of Owings Mills,Md.), are added and mixed well to dissolve. All of the OSV blend is thenadded. The containers are rinsed out 4 to 5 times with the oil blend toassure complete transfer of the vitamins. The carrageenan (distributedby FMC of Rockland, Me.) and the caseinate are added. The slurry ismixed well to disperse the protein. The PIF slurry is held up to sixhours at 60-65° C. under moderate agitation until used.

The stock protein in water slurry (PIW) is prepared by adding therequired amount of water to a blend tank. The water is held undermoderate agitation and brought up to 76-82° C. The required amount ofcaseinate is added to the water under high agitation and mixed on highuntil the protein is fully dispersed. The protein slurry is allowed tocool to 54-60° C. before proceeding. Once cooled the required amount ofwhey protein is added and mixed well until fully dispersed/dissolved.The PIW slurry is held up to two hours at 54-60° C. until used.

The stock mineral solution (MIN) is prepared by adding the requiredamount of water to a blend tank and heated to 60-68° C. The cellulosegum blend (distributed by FMC of Newark, Del.) is added to the water andheld under moderate agitation for a minimum of five minutes beforeproceeding. The calcium HMB is added and agitated for a minimum of fiveminutes before proceeding. The mineral salts magnesium chloride,potassium chloride, potassium citrate, potassium iodide and dipotassiumphosphate are added one at a time with mixing between each addition toensure the minerals dissolved. The completed MIN solution is held at54-65° C. under low to moderate agitation until used.

The final blend is prepared by adding the specified amount of PIW slurryto a blend tank and heated under agitation to 54-60° C. The specifiedamount of PIF slurry is added to the tank and mixed well. The specifiedamount of MIN solution is added to the blend and mixed well. Thespecified amount of sodium chloride is added to the blend and mixedwell. The specified amount of sucrose is added to the blend and mixedwell to dissolve. The tricalcium phosphate is added to the blend andmixed well to disperse. The specified amount of additional water isadded to the blend and mixed well. The completed final blend is heldunder continuous agitation at 54-60° C. If necessary, the pH is adjustedto 6.45-6.8 with 1N KOH.

After waiting for a period of not less than one minute nor greater thantwo hours, the blend slurry is subjected to deaeration,ultra-high-temperature treatment, and homogenization. The blended slurryis heated to a temperature from about 68° C. to about 74° C. anddeaerated under vacuum. The heated slurry is then emulsified at 900 to1100 psig. After emulsification, the slurry is heated from about 120° C.to about 122° C. and then heated to a temperature of about 149° C. toabout 150° C. The slurry is passed through a flash cooler to reduce thetemperature to from about 120° C. to about 122° C. and then through aplate cooler to reduce the temperature to from about 74° C. to about 79°C. The slurry is then homogenized at 3900 to 4100/400 to 600 psig. Theslurry is held at about 74° C. to about 85° C. for 16 seconds and thencooled to 1° C. to about 6° C. At this point, samples are taken formicrobiological and analytical testing. The mixture is held underagitation.

Standardization proceeds as follows. The stock vitamin solution (WSV) isprepared by heating the specified amount of water to 48-60° C. in ablend tank. Potassium citrate, UTM/TM premix (distributed by Fortitechof Schenectady, N.Y.), WSV premix, m-inositol, taurine, L-carnitine andcholine chloride are each added to the solution in the order listed andallowed to mix well to dissolve or disperse each ingredient. 14.2 kg ofthe vitamin solution is added to the processed mix tank.

The stock vanilla solution is prepared by adding the specified amount ofwater to a blend tank. The specified amount of vanilla (distributed byGivaudan Roure of Cincinnati, Ohio) is added to the water and mixedwell. 18.5 kg of vanilla solution is added to the processed mix tank andmixed well.

The stock ascorbic acid solution is prepared by adding the requiredamount of water to a blend tank. The specified amount of ascorbic acidis added and mixed well to dissolve. The specified amount of 45% KOH isadded and mixed well. 8.4 kg of ascorbic acid solution is added to themix tank and mixed well.

The final mix is diluted to the final total solids by adding 92.5 kg ofwater and mixed well. Product is filed into suitable containers prior toterminal (retort) sterilization.

Example VIII Composition of a Complete Nutritional Supplement

Table 4 presents a bill of materials for manufacturing 1,000 kg of atypical vanilla flavored meal replacement liquid. A detailed descriptionof its manufacture follows.

TABLE 4 Bill of Materials for Vanilla Liquid Nutritional IngredientQuantity per 1,000 kg Water QS Corn Syrup 33 kg Maltodextrin 28 kgSucrose 19.4 kg Caseinate 8.7 kg Calcium HMB monohydrate 5.7 kg HighOleic Safflower Oil 4.1 kg Canola Oil 4.1 kg Soy Protein 3.7 kg WheyProtein 3.2 kg Caseinate 2.9 kg Corn Oil 2.0 kg Tricalcium Phosphate 1.4kg Potassium Citrate 1.3 kg Magnesium Phosphate 952 gm Lecithin 658 gmMagnesium chloride 558 gm Vanilla Flavor 544 gm Sodium Chloride 272 gmCarrageenan 227 gm Choline chloride 218 gm UTM/TM Premix 165 gmPotassium Chloride 146 gm Ascorbic Acid 145 gm Sodium Citrate 119 gmPotassium Hydroxide 104 gm Lutein (5%) 46 gm WSV Premix 33 gm Vit DEKPremix 29 gm Vitamin A 3.7 gm Potassium Iodide 86 mcg WSV premix(per gpremix): 375 mg/g niacinamide, 242 mg/g calcium pantothenate, 8.4 gm/gfolic acid, 62 mg/g thiamine chloride hydrochloride, 48.4 gm/griboflavin, 59.6 mg/g pyridoxine hydrochloride, 165 mcg/g cyanocobalaminand 7305 mcg/g biotin Vitamin DEK premix (per g premix): 8130 IU/gvitamin D₃, 838 IU/g vitamin E, 1.42 mg/g vitamin K₁ UTM/TM premix (perg premix): 45.6 mg/g zinc, 54 mg/g iron, 15.7 manganese, 6.39 mg/gcopper, 222 mcg/g selenium, 301 mcg/g chromium and 480 mcg/g molybdenium

The liquid meal replacement products of the present invention aremanufactured by preparing three slurries that are blended together, heattreated, standardized, packaged and sterilized.

A carbohydrate/mineral slurry is prepared by first heating the requiredamount of water to a temperature of from about 65° C. to about 71° C.with agitation. The required amount of calcium HMB is added and agitatedfor a minimum of 5 minutes. With agitation, the required amount ofpotassium citrate and ultra trace mineral/trace mineral (UTM/TM) premix(distributed by Fortitech, Schnectady, N.Y.) is added. The slurry isgreenish yellow in color. Agitation is maintained until the minerals arecompletely dispersed. With agitation, the required amounts of thefollowing minerals are then added: magnesium chloride, potassiumchloride, sodium chloride, sodium citrate, potassium iodide, magnesiumphosphate and tricalcium phosphate. Next, the maltodextrin distributedby Grain Processing Corporation, Muscataine, Iowa, U.S.A., sucrose andcorn syrup are added to slurry under high agitation, and are allowed todissolve. The completed carbohydrate/mineral slurry is held withagitation at a temperature from about 65° C. to about 71° C. for notlonger than eight hours until it is blended with the other slurries.

A protein in fat slurry (PIF) is prepared by combining and heating therequired amounts of high oleic safflower oil and canola oil to atemperature from about 40.5° C. to about 49° C. with agitation. Withagitation, the required amounts of free lutein from Kemin Foods of DesMoines, Iowa is added. Agitate for a minimum of 15 minutes. Add thefollowing ingredients are added to the heated oil: lecithin (distributedby Central Soya Company, Fort Wayne, Ind.), vitamin A, and Vitamin D, E,K premix (distributed by Vitamins Inc., Chicago, Ill.). The requiredamount of carrageenan is dry blended with the required amount of wheyprotein and add to the agitating lipid mixture and allowed to agitatefor a minimum of 10 minutes. The required amount of soy protein is addedto the blend slowly to assure proper mixing. The completed oil/proteinslurry is held under moderate agitation at a temperature from about 40°C. to about 43° C. for a period of no longer than two hours until it isblended with the other slurries.

A protein in water slurry is prepared by first heating about requiredamount of water to a temperature of about 40° C. with agitation. Thecaseinate is added and the slurry is agitated well until the caseinateis completely dispersed. With continued agitation, the slurry is slowlywarmed to 60° C. to 65° C. The slurry is held for no longer than twelvehours until it is blended with the other slurries.

The batch is assembled by blending required amount of protein slurrywith required amount of the carbohydrate/mineral slurry and allowed toagitate for 10 minutes. With agitation, the required amount of theoil/protein slurry is added and agitate for at least 10 minutes. The pHof the blended batch is adjusted to a pH of 6.66 to 6.75 with 1Npotassium hydroxide.

After waiting for a period of not less than one minute nor greater thantwo hours, the blend slurry is subjected to deaeration,ultra-high-temperature treatment, and homogenization. The blended slurryis heated to a temperature from about 71° C. to about 82° C. anddeareated under vacuum. The heated slurry is then emulsified through asingle stage homogenizer at 900 to 1100 psig. After emulsification, theslurry is heated from about 99° C. to about 110° C. and then heated to atemperature of about 146° C. for about 5 seconds. The slurry is passedthrough a flash cooler to reduce the temperature to from about 99° C. toabout 110° C. and then through a plate cooler to reduce the temperatureto from about 71° C. to about 76° C. The slurry is then homogenized at3900 to 4100/400 to 600 psig. The slurry is held at about 74° C. toabout 80° C. for 16 seconds and then cooled to 1° C. to about 7° C. Atthis point, samples are taken for microbiological and analyticaltesting. The mixture is held under agitation.

A water soluble vitamin (WSV) solution is prepared separately and addedto the processed blended slurry.

The vitamin solution is prepared by adding the following ingredients to9.4 kg of water with agitation: WSV premix (distributed by J.B.Laboratories, Holland, Mich.), vitamin C, choline chloride, L-carnitine,taurine, inositiol, folic acid, pyridoxine hydrochloride andcyanocobalamin. The required amount of 45% potassium hydroxide slurry isadded to bring the pH to between 7 and 10.

Based on the analytical results of the quality control tests, anappropriate amount of water is added to the batch with agitation toachieve the desired total solids. Additionally, 8.8 kg of vitaminsolution is added to the diluted batch under agitation.

The product pH may be adjusted to achieve optimal product stability. Thecompleted product is then placed in suitable containers and subjected toterminal sterilization.

Example IX Composition of a Beverage

To produce a 1000 kg batch of ready-to-drink beverage, 987.31 kg ofwater is placed in a vessel fitted with an agitator. At ambienttemperature, the required amount of potassium benzoate is added andallowed to completely dissolve. The required amount of calcium HMB isadded and allowed to completely dissolve. The following ingredients arethen added in the order listed. Each ingredient is completely dissolvedbefore the next ingredient is added.

TABLE 5 Ready-to-drink beverage Potassium benzoate 0.30 kg Calcium HMBmonohydrate  5.7 kg Potassium Citrate 0.15 kg Citric Acid 2.89 kg LacticAcid 1.41 kg Aspartame 0.55 kg Calcium Glycerophosphate 6.06 kg ColoringAgents 0.0019 kg  Natural and artificial flavors 1.00 kg Ascorbic acid0.33 kg

The ascorbic acid was added just before filling into 12-oz. aluminumcans. The beverages may be carbonated prior to filling into aluminumcans. The solution is de-aerated and then transferred to a“carbo-cooler” where it is cooled and carbonated to approximately 2.5volumes of carbon dioxide.

Example X Composition of an Electrolyte Replacement Product

The following example explains how to manufacture a ready-to-drinkrehydration solution. The ORS had the composition outlined in Table 6.

TABLE 6 Ready-to-drink Rehydration Solution Ingredient Quantity per 454kg Water 437 kg Dextrose, Monohydrate 10 kg Fructose 2.4 kg Citric Acid1.2 kg Sodium Chloride 0.937 kg Potassium Citrate 1 kg Sodium Citrate492.0 g Calcium HMB monohydrate 5.7 kg Fruit Flavor 226.8 g ZincGluconate 80.62 g Sucralose 179.2 g Acesulfame Potassium 38.1 g Yellow#6 7.2 g

Weigh out the required amount of filtered water and add to blend tank.Heat the water to 43-54° C., with moderate agitation. While maintainingmoderate agitation, the calcium HMB is added and allowed to mix for aminimum of 5 minutes. With continued moderate agitation add the requiredamount of dextrose. Agitate until dissolved. Add the required amount offructose. Agitate until dissolved. Add the required amount of thefollowing ingredients, in the order listed, to the dextrose/fructoseblend and agitate until dissolved: zinc gluconate, sodium citrate,sodium chloride, potassium citrate, and citric acid. Add the requiredamount of sucralose (distributed by McNeil Specialty Products Company ofNew Brunswick, N.J.) and acesulfame potassium (distributed as Sunsett®by Hoechst Food Ingredients of Somerset, N.J.) and agitate untildissolved. Add the yellow #6 and the fruit punch flavor to the batchuntil dissolved. Cool the blend to 1.1-7.2° C. and hold with lowagitation. Fill the required number of one liter plastic bottles, applythe foil heat seal to the bottle opening, and retort to food gradesterility standards.

Alternatively, the cooled blend is encapsulated within a sealablefreezable packaging material and sealed such as by heat sealing. Asingle dose of rehydration solution is packaged in a hermetically sealedfreezable pouch. Various types of packaging materials which can be usedto practice the invention, such as that used in traditional freezerpops, would be readily apparent to the skilled artisan. The wrappingmaterial is preferably a type which will allow markings, such as productidentification, ingredients, etc., to be placed on the exterior surfacethereof. The rehydration formulation is shipped and stored, preferablyin multiple units thereof, in this condition. It is contemplated thatmultiple units or freezer pops will be packaged together for purposes ofcommercialization.

Prior to administration, a package of liquid rehydration solution isfrozen. Following freezing, the package is opened and the contentsthereof eaten. Since the frozen rehydration formulation will normally beadministered at ambient temperatures, the amount of rehydration liquidcontained in each package is preferably an amount which can be consumedin its entirely while still in the frozen state. Preferably 20-35ounces, more preferably 2.0 to 2.5 ounces per package. In a particularlypreferred embodiment, 2.1 ounces of sterile rehydration solution isencapsulated within an rectangular, e.g., 1“.times.8,” freezable wrappermaterial. Clear plastic wrapper material is preferred.

Example XI Effect of HMB on Protein Synthesis (vs. Degradation) inSkeletal Muscle

Materials. Materials and animals were obtained as per Example 1.

Methodologies. Protein synthesis was measured by the incorporation ofL-[4-3H]phenylalanine during a 2-hour period in which isolatedgastrocnemius muscles were incubated at 37° C. in RPMI 1640 withoutphenol red and saturated with O2/CO2 (19:1). After incubation muscleswere rinsed in nonradioactive medium, blotted dry, and homogenized in 4mL 2% perchloric acid. The rate of protein synthesis was calculated bydividing the protein-bound radioactivity by the acid-soluble (unbound)material.

For protein degradation assays animals from the same group as used tomeasure protein synthesis were given i.p. 0.4 mmol/LL-[4-3H]phenylalanine in PBS (100 μl) 24 hours prior to the assay.Isolated gastrocnemius muscles were extensively washed with PBS and RPMI1640 before measuring the release of radioactivity into RPMI 1640 over a2-hour period. The protein-bound activity was determined by homogenizingthe muscles in 2% perchloric acid and determining the non-acid-solubleradioactivity (radioactivity in the precipitate). The rate of proteindegradation was calculated by dividing the amount of [³H]phenylalanineradioactivity released into the incubation medium during the 2-hourincubation period by the specific activity of protein-bound [³H]phenylalanine.

Western Blot analysis and statistical treatments were as described inExample 1.

Results. The protein degradation in these muscles was measured bytyrosine released per gram of muscle over 2 hours, taking intoconsideration the variation in muscle sizes (FIG. 4B). At the doseschosen HMB was as effective as EPA and there did not seem to be asynergistic effect of the combination, however, the combination of HMBwith EPA may permit lower doses of each ingredient without loss ofefficacy. Body composition analysis (Table 1) indicated that HMB causeda significant increase in the nonfat carcass mass without an effect onadipose tissue.

TABLE 1 Body composition analysis of mice bearing the MAC16 tumortreated with HMB for 5 days HMB (g/kg) Water (%) Fat (%) Nonfat (%) 070.8 ± 2.7 3.8 ± 1.3 25.4 ± 1.9  0.125 65.5 ± 0.9 3.3 ± 1.6 31.2 ± 1.5*0.25 66.5 ± 2.2 4.4 ± 1.3 29.2 ± 1.9* 0.5 67.2 ± 2.3 3.8 ± 1.2 29.0 ±1.4* *P < 0.01 from control group receiving 0 g/kg HMB.

EPA has been shown to attenuate the increase in protein degradation inskeletal muscle of mice bearing the MAC16 tumor but has no effect on thedepression of protein synthesis (Beck, et al. Cancer Res. 1991;284:206-10.). In contrast, HMB, when evaluated at two dose levels (0.25and 2.5 g/kg) in the present experiment not only attenuated proteindegradation but also significantly increased protein synthesis ingastrocnemius muscle of mice bearing the MAC16 tumor when compared withcontrol animals receiving PBS. This is shown in FIG. 14A, wheresynthesis (dark-shaded columns) increases compared to control anddegradation (unshaded columns) decreases compared to control. Thisopposite effect resulted in an increase in the ratio of proteinsynthesis to protein degradation in muscle (FIG. 14B) by 14-fold withHMB at 0.25 g/kg and by 32-fold at 2.5 g/kg.

Lean muscle status is always a dynamic system of synthesis anddegradation. This study shows that HMB can affect both processesfavorably in wasting patients to increase overall lean mass. HMB iscapable not only of attenuating protein degradation in skeletal muscle,but also stimulates protein synthesis resulting in an increase in thenonfat carcass mass. This is particularly important in wasting diseasestates like cachexia and AIDS that manifest themselves as loss of leanmuscle loss.

1. A composition comprising: a. from about 2 to 10 grams per litercalcium beta-hydroxy-beta-methylbutyrate; b. at least 1 gram per literof omega-3 fatty acids; c. from about 1 to about 8 grams per litercarnitine; d. from about 1 to about 25 grams per liter FOS; and e. anamino nitrogen source enriched with large neutral amino acids, whereinthe amino nitrogen source comprises from about 10 to 60 wt/wt % largeneutral amino acids; and wherein the composition is substantiallylacking in free amino acids.
 2. The composition according to claim 1wherein the omega-3 fatty acids are selected from the group consistingof alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid,docosapentaenoic acid and docohexaenoic acid.
 3. The compositionaccording to claim 2 wherein the omega-3 fatty acids areeicosapentaenoic acid.
 4. The composition according to claim 1comprising less than 0.4 grams per serving of free amino acids.
 5. Thecomposition according to claim 1 further comprising a nutrient selectedfrom the group consisting of vitamins, minerals, and trace minerals. 6.The composition according to claim 1 further comprising an antioxidantselected from the group consisting of beta-carotene, vitamin E, vitaminC, and selenium.
 7. The composition according to claim 1 wherein thecomposition is administered to a human.
 8. The composition according toclaim 1 wherein the composition is selected from the group consisting ofdietary supplement, meal replacement, nutritional bar, chew or bite andbeverage.
 9. The composition according to claim 1 further comprisingomega-6 fatty acids.
 10. The composition according to claim 9 whereinthe weight ratio of omega-3 fatty acids to omega-6 fatty acids is about0.1 to 3.0.
 11. The composition according to claim 1 wherein the largeneutral amino acids are selected from the group consisting of valine,isoleucine, leucine, threonine, tyrosine, and phenylalanine.
 12. Thecomposition according to claim 1 further comprising a fiber.
 13. Thecomposition according to claim 12 wherein the fiber is selected from thegroup consisting of gum arabic, hydrolyzed carboxymethylcellulose, guargum, pectin, oat glucan, barley glucan, psyllium, hull oat fiber, peahull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber,cellulose, and corn bran.
 14. The composition according to claim 1further comprising a stabilizer.
 15. The composition according to claim14 wherein the stabilizer is selected from the group consisting ofxanthan gum, guar gum, gum arabic, gum ghatti, gum karaya, gumtracacanth, agar, fucellaran, gellan gum, locust bean gum, pectin, oatglucan, barley glucan, psyllium, gelatin, microcrystalline cellulose,sodium carboxymethylcelluose, methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, diacetyl tartaric acid esters ofmonoglycerides and diglycerides, dextran, fructooligosaccharides, andmixtures thereof.
 16. The composition according to claim 1 furthercomprising an artificial sweetener.
 17. The composition according toclaim 16 wherein the artificial sweetener is selected from the groupconsisting of saccharin, sucralose, and acesulfane-K.
 18. A method oftreating or preventing a disease condition in a patient comprisingadministering to the patient the composition of claim
 1. 19. The methodaccording to claim 18 wherein the disease condition is selected from thegroup consisting of cancer, cachexia, age-associated wasting, wastingassociated with long-term hospitalization, human immunodeficiencyvirus/acquired immunodeficiency syndrome, arthritis, trauma, liverdisease, Crohn's disease, inflammatory bowel disease, renalinsufficiency and chronic obstructive pulmonary disease.
 20. The methodaccording to claim 19 wherein the disease condition is cachexia.